Combinatorial DNA library for producing modified N-glycans in lower eukaryotes

ABSTRACT

The present invention relates to eukaryotic host cells having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities such as those involved in glycosylation to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified oligosaccharides are created or selected. N-glycans made in the engineered host cells have a Man 5 GlcNAc 2  core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar transporters and mannosidases, to yield human-like glycoproteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/156,804, filed Jun. 9, 2011 which is a continuation of U.S.application Ser. No. 12/291,373 filed Nov. 7, 2008, now U.S. Pat. No.8,067,551 which is a continuation of U.S. application Ser. No.10/371,877, filed Feb. 20, 2003, now U.S. Pat. No. 7,449,308, which is acontinuation-in-part of U.S. application Ser. No. 09/892,591, filed Jun.27, 2001, now U.S. Pat. No. 7,029,872, which claims the benefit of U.S.Provisional Application Ser. No. 60/214,358, filed Jun. 28, 2000; U.S.Provisional Application Ser. No. 60/215,638, filed Jun. 30, 2000, andU.S. Provisional Application Ser. No. 60/279,997, filed Mar. 30, 2001;each of which is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was funded, at least in part, with a government grantfrom the National Institutes of Health (NHI Phase I Grant No.1R43GM66690-1) and a grant from the Department of Commerce, NIST-ATPCooperative Agreement Number 70NANB2H3046. The United States Governmentmay therefore have certain rights in this invention.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions by whichnon-human eukaryotic host cells, such as fungi or other eukaryoticcells, can be genetically modified to produce glycosylated proteins(glycoproteins) having patterns of glycosylation similar to those ofglycoproteins produced by animal cells, especially human cells, whichare useful as human or animal therapeutic agents.

BACKGROUND OF THE INVENTION

Glycosylation Pathways in Humans and Lower Eukaryotes

After DNA is transcribed and translated into a protein, furtherpost-translational processing involves the attachment of sugar residues,a process known as glycosylation. Different organisms produce differentglycosylation enzymes (glycosyltransferases and glycosidases), and havedifferent substrates (nucleotide sugars) available, so that theglycosylation patterns as well as composition of the individualoligosaccharides, even of the same protein, will be different dependingon the host system in which the particular protein is being expressed.Bacteria typically do not glycosylate proteins, and if so only in a veryunspecific manner (Moens and Vanderleyden, 1997 Arch Microbiol.168(3):169-175). Lower eukaryotes such as filamentous fungi and yeastadd primarily mannose and mannosylphosphate sugars. The resulting glycanis known as a “high-mannose” type glycan or a mannan. Plant cells andinsect cells (such as Sf9 cells) glycosylate proteins in yet anotherway. By contrast, in higher eukaryotes such as humans, the nascentoligosaccharide side chain may be trimmed to remove several mannoseresidues and elongated with additional sugar residues that typically arenot found in the N-glycans of lower eukaryotes. See, e.g., R. K.Bretthauer, et al. Biotechnology and Applied Biochemistry, 1999, 30,193-200; W. Martinet, et al. Biotechnology Letters, 1998, 20, 1171-1177;S. Weikert, et al. Nature Biotechnology, 1999, 17, 1116-1121; M.Malissard, et al. Biochemical and Biophysical Research Communications,2000, 267, 169-173; Jarvis, et al., Current Opinion in Biotechnology,1998, 9:528-533; and M. Takeuchi, 1 Trends in Glycoscience andGlycotechnology, 1997, 9, S29-S35.

Synthesis of a mammalian-type oligosaccharide structure begins with aset of sequential reactions in the course of which sugar residues areadded and removed while the protein moves along the secretory pathway inthe host organism. The enzymes which reside along the glycosylationpathway of the host organism or cell determine the resultingglycosylation patterns of secreted proteins. Thus, the resultingglycosylation pattern of proteins expressed in lower eukaryotic hostcells differs substantially from the glycosylation pattern of proteinsexpressed in higher eukaryotes such as humans and other mammals(Bretthauer, 1999). The structure of a typical fungal N-glycan is shownin FIG. 1A.

The early steps of human glycosylation can be divided into at least twodifferent phases: (i) lipid-linked Glc₃Man₉GlcNAc₂ oligosaccharides areassembled by a sequential set of reactions at the membrane of theendoplasmic reticulum (ER) and (ii) the transfer of this oligosaccharidefrom the lipid anchor dolichyl pyrophosphate onto de novo synthesizedprotein. The site of the specific transfer is defined by an asparagine(Asn) residue in the sequence Asn-Xaa-Ser/Thr where Xaa can be any aminoacid except proline (Gavel, 1990). Further processing by glucosidasesand mannosidases occurs in the ER before the nascent glycoprotein istransferred to the early Golgi apparatus, where additional mannoseresidues are removed by Golgi specific alpha (α)-1,2-mannosidases.Processing continues as the protein proceeds through the Golgi. In themedial Golgi, a number of modifying enzymes, includingN-acetylglucosaminyl Transferases (GnTI, GnTII, GnTIII, GnTIV and GnTV),mannosidase II and fucosyltransferases, add and remove specific sugarresidues. Finally, in the trans-Golgi, galactosyltranferases (GalT) andsialyltransferases (ST) produce a glycoprotein structure that isreleased from the Golgi. It is this structure, characterized by bi-,tri- and tetra-antennary structures, containing galactose, fucose,N-acetylglucosamine and a high degree of terminal sialic acid, thatgives glycoproteins their human characteristics. The structure of atypical human N-glycan is shown in FIG. 1B.

In nearly all eukaryotes, glycoproteins are derived from a commonlipid-linked oligosaccharide precursorGlc₃Man₉GlcNAc₂-dolichol-pyrophosphate. Within the endoplasmicreticulum, synthesis and processing of dolichol pyrophosphate boundoligosaccharides are identical between all known eukaryotes. However,further processing of the core oligosaccharide by fungal cells, e.g.,yeast, once it has been transferred to a peptide leaving the ER andentering the Golgi, differs significantly from humans as it moves alongthe secretory pathway and involves the addition of several mannosesugars.

In yeast, these steps are catalyzed by Golgi residingmannosyltransferases, like Och1p, Mnt1p and Mnn1p, which sequentiallyadd mannose sugars to the core oligosaccharide. The resulting structureis undesirable for the production of human-like proteins and it is thusdesirable to reduce or eliminate mannosyltransferase activity. Mutantsof S. cerevisiae, deficient in mannosyltransferase activity (for exampleoch1 or mnn9 mutants) have been shown to be non-lethal and displayreduced mannose content in the oligosaccharide of yeast glycoproteins.Other oligosaccharide processing enzymes, such as mannosylphosphatetransferase, may also have to be eliminated depending on the host'sparticular endogenous glycosylation pattern.

Sugar Nucleotide Precursors

The N-glycans of animal glycoproteins typically include galactose,fucose, and terminal sialic acid. These sugars are not found onglycoproteins produced in yeast and filamentous fungi. In humans, thefull range of nucleotide sugar precursors (e.g. UDP-N-acetylglucosamine,UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose,GDP-fucose, etc.) are synthesized in the cytosol and transported intothe Golgi, where they are attached to the core oligosaccharide byglycosyltransferases. (Sommers and Hirschberg, 1981 J. Cell Biol. 91(2):A406-A406; Sommers and Hirschberg 1982 J. Biol. Chem. 257(18): 811-817;Perez and Hirschberg 1987 Methods in Enzymology 138: 709-715).

Glycosyl transfer reactions typically yield a side product which is anucleoside diphosphate or monophosphate. While monophosphates can bedirectly exported in exchange for nucleoside triphosphate sugars by anantiport mechanism, diphosphonucleosides (e.g. GDP) have to be cleavedby phosphatases (e.g. GDPase) to yield nucleoside monophosphates andinorganic phosphate prior to being exported. This reaction is importantfor efficient glycosylation; for example, GDPase from Saccharomycescerevisiae (S. cerevisiae) has been found to be necessary formannosylation. However that GDPase has 90% reduced activity toward UDP(Berninsone et al., 1994 J. Biol. Chem. 269(1):207-211). Lowereukaryotes typically lack UDP-specific diphosphatase activity in theGolgi since they do not utilize UDP-sugar precursors for Golgi-basedglycoprotein synthesis. Schizosaccharomyces pombe, a yeast found to addgalactose residues to cell wall polysaccharides (from UDP-galactose) hasbeen found to have specific UDPase activity, indicating the potentialrequirement for such an enzyme (Berninsone et al., 1994). UDP is knownto be a potent inhibitor of glycosyltransferases and the removal of thisglycosylation side product may be important to preventglycosyltransferase inhibition in the lumen of the Golgi (Khatara etal., 1974). See Berninsone, P., et al. 1995. J. Biol. Chem. 270(24):14564-14567; Beaudet, L., et al. 1998 Abc Transporters: Biochemical,Cellular, and Molecular Aspects. 292: 397-413.

Sequential Processing of N-Glycans by Compartmentalized EnzymeActivities

Sugar transferases and glycosidases (e.g., mannosidases) line the inner(luminal) surface of the ER and Golgi apparatus and thereby provide a“catalytic” surface that allows for the sequential processing ofglycoproteins as they proceed through the ER and Golgi network. Themultiple compartments of the cis, medial, and trans Golgi and thetrans-Golgi Network (TGN), provide the different localities in which theordered sequence of glycosylation reactions can take place. As aglycoprotein proceeds from synthesis in the ER to full maturation in thelate Golgi or TGN, it is sequentially exposed to different glycosidases,mannosidases and glycosyltransferases such that a specific carbohydratestructure may be synthesized. Much work has been dedicated to revealingthe exact mechanism by which these enzymes are retained and anchored totheir respective organelle. The evolving picture is complex but evidencesuggests that stem region, membrane spanning region and cytoplasmictail, individually or in concert, direct enzymes to the membrane ofindividual organelles and thereby localize the associated catalyticdomain to that locus (see, e.g., Gleeson, P. A. (1998) Histochem. CellBiol. 109, 517-532).

In some cases, these specific interactions were found to function acrossspecies. For example, the membrane spanning domain of α2,6-ST from rats,an enzyme known to localize in the trans-Golgi of the animal, was shownto also localize a reporter gene (invertase) in the yeast Golgi(Schwientek, 1995). However, the very same membrane spanning domain aspart of a full-length α2,6-ST was retained in the ER and not furthertransported to the Golgi of yeast (Krezdorn, 1994). A full length GalTfrom humans was not even synthesized in yeast, despite demonstrably hightranscription levels. In contrast, the transmembrane region of the samehuman GalT fused to an invertase reporter was able to directlocalization to the yeast Golgi, albeit it at low production levels.Schwientek and co-workers have shown that fusing 28 amino acids of ayeast mannosyltransferase (MNT1), a region containing a cytoplasmictail, a transmembrane region and eight amino acids of the stem region,to the catalytic domain of human GalT are sufficient for Golgilocalization of an active GalT. Other galactosyltransferases appear torely on interactions with enzymes resident in particular organellesbecause, after removal of their transmembrane region, they are stillable to localize properly.

Improper localization of a glycosylation enzyme may prevent properfunctioning of the enzyme in the pathway. For example, Aspergillusnidulans, which has numerous α-1,2-mannosidases (Eades and Hintz, 2000Gene 255(1):25-34), does not add GlcNAc to Man₅GlcNAc₂ when transformedwith the rabbit GnTI gene, despite a high overall level of GnTI activity(Kalsner et al., 1995). GnTI, although actively expressed, may beincorrectly localized such that the enzyme is not in contact with bothof its substrates: UDP-GlcNAc and a productive Man₅GlcNAc₂ substrate(not all Man₅GlcNAc₂ structures are productive; see below).Alternatively, the host organism may not provide an adequate level ofUDP-GlcNAc in the Golgi or the enzyme may be properly localized butnevertheless inactive in its new environment. In addition, Man₅GlcNAc₂structures present in the host cell may differ in structure fromMan₅GlcNAc₂ found in mammals. Maras and coworkers found that about onethird of the N-glycans from cellobiohydrolase I (CBHI) obtained from T.reesei can be trimmed to Man₅GlcNAc₂ by A. saitoi 1,2 mannosidase invitro. Fewer than 1% of those N-glycans, however, could serve as aproductive substrate for GnTI. The mere presence of Man₅GlcNAc₂,therefore, does not assure that further processing to Man₅GlcNAc₂ can beachieved. It is formation of a productive, GnTI-reactive Man₅GlcNAc₂structure that is required. Although Man₅GlcNAc₂ could be produced inthe cell (about 27 mol %), only a small fraction could be converted toMan₅GlcNAc₂ (less than about 5%, see Chiba WO 01/14522).

To date, there is no reliable way of predicting whether a particularheterologously expressed glycosyltransferase or mannosidase in a lowereukaryote will be (1), sufficiently translated (2), catalytically activeor (3) located to the proper organelle within the secretory pathway.Because all three of these are necessary to affect glycosylationpatterns in lower eukaryotes, a systematic scheme to achieve the desiredcatalytic function and proper retention of enzymes in the absence ofpredictive tools, which are currently not available, would be desirable.

Production of Therapeutic Glycoproteins

A significant number of proteins isolated from humans or animals arepost-translationally modified, with glycosylation being one of the mostsignificant modifications. An estimated 70% of all therapeutic proteinsare glycosylated and thus currently rely on a production system (i.e.,host cell) that is able to glycosylate in a manner similar to humans.Several studies have shown that glycosylation plays an important role indetermining the (1) immunogenicity, (2) pharmacokinetic properties, (3)trafficking, and (4) efficacy of therapeutic proteins. It is thus notsurprising that substantial efforts by the pharmaceutical industry havebeen directed at developing processes to obtain glycoproteins that areas “humanoid” or “human-like” as possible. To date, most glycoproteinsare made in a mammalian host system. This may involve the geneticengineering of such mammalian cells to enhance the degree of sialylation(i.e., terminal addition of sialic acid) of proteins expressed by thecells, which is known to improve pharmacokinetic properties of suchproteins. Alternatively, one may improve the degree of sialylation by invitro addition of such sugars using known glycosyltransferases and theirrespective nucleotide sugars (e.g., 2,3-sialyltransferase and CMP-sialicacid).

While most higher eukaryotes carry out glycosylation reactions that aresimilar to those found in humans, recombinant human proteins expressedin the above mentioned host systems invariably differ from their“natural” human counterpart (Raju, 2000). Extensive development work hasthus been directed at finding ways to improve the “human character” ofproteins made in these expression systems. This includes theoptimization of fermentation conditions and the genetic modification ofprotein expression hosts by introducing genes encoding enzymes involvedin the formation of human-like glycoforms (Werner, 1998; Weikert, 1999;Andersen, 1994; Yang, 2000). Inherent problems associated with allmammalian expression systems have not been solved.

Fermentation processes based on mammalian cell culture (e.g., CHO,murine, or human cells), for example, tend to be very slow (fermentationtimes in excess of one week are not uncommon), often yield low producttiters, require expensive nutrients and cofactors (e.g., bovine fetalserum), are limited by programmed cell death (apoptosis), and often donot enable expression of particular therapeutically valuable proteins.More importantly, mammalian cells are susceptible to viruses that havethe potential to be human pathogens and stringent quality controls arerequired to assure product safety. This is of particular concern as manysuch processes require the addition of complex and temperature sensitivemedia components that are derived from animals (e.g., bovine calfserum), which may carry agents pathogenic to humans such as bovinespongiform encephalopathy (BSE) prions or viruses. Moreover, theproduction of therapeutic compounds is preferably carried out in awell-controlled sterile environment. An animal farm, no matter howcleanly kept, does not constitute such an environment, thus constitutingan additional problem in the use of transgenic animals for manufacturinghigh volume therapeutic proteins.

Most, if not all, currently produced therapeutic glycoproteins aretherefore expressed in mammalian cells and much effort has been directedat improving (i.e., “humanizing”) the glycosylation pattern of theserecombinant proteins. Changes in medium composition as well as theco-expression of genes encoding enzymes involved in human glycosylationhave been successfully employed (see, for example, Weikert, 1999).

Glycoprotein Production Using Eukaryotic Microorganisms

The lack of a suitable mammalian expression system is a significantobstacle to the low-cost and safe production of recombinant humanglycoproteins for therapeutic applications. It would be desirable toproduce recombinant proteins similar to their mammalian, e.g., human,counterparts in lower eukaryotes (fungi and yeast). Production ofglycoproteins via the fermentation of microorganisms would offernumerous advantages over existing systems. For example,fermentation-based processes may offer (a) rapid production of highconcentrations of protein; (b) the ability to use sterile,well-controlled production conditions; (c) the ability to use simple,chemically defined (and protein-free) growth media; (d) ease of geneticmanipulation; (e) the absence of contaminating human or animal pathogenssuch as viruses; (f) the ability to express a wide variety of proteins,including those poorly expressed in cell culture owing to toxicity etc.;and (g) ease of protein recovery (e.g. via secretion into the medium).In addition, fermentation facilities for yeast and fungi are generallyfar less costly to construct than cell culture facilities. Although thecore oligosaccharide structure transferred to a protein in theendoplasmic reticulum is basically identical in mammals and lowereukaryotes, substantial differences have been found in the subsequentprocessing reactions which occur in the Golgi apparatus of fungi andmammals. In fact, even amongst different lower eukaryotes there exist agreat variety of glycosylation structures. This has historicallyprevented the use of lower eukaryotes as hosts for the production ofrecombinant human glycoproteins despite otherwise notable advantagesover mammalian expression systems.

Therapeutic glycoproteins produced in a microorganism host such as yeastutilizing the endogenous host glycosylation pathway differ structurallyfrom those produced in mammalian cells and typically show greatlyreduced therapeutic efficacy. Such glycoproteins are typicallyimmunogenic in humans and show a reduced half-life (and thusbioactivity) in vivo after administration (Takeuchi, 1997). Specificreceptors in humans and animals (i.e., macrophage mannose receptors) canrecognize terminal mannose residues and promote the rapid clearance ofthe foreign glycoprotein from the bloodstream. Additional adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity.

Yeast and filamentous fungi have both been successfully used for theproduction of recombinant proteins, both intracellular and secreted(Cereghino, J. L. and J. M. Cregg 2000 FEMS Microbiology Reviews 24(1):45-66; Harkki, A., et al. 1989 Bio-Technology 7(6): 596; Berka, R. M.,et al. 1992 Abstr. Papers Amer. Chem. Soc. 203: 121-BIOT; Svetina, M.,et al. 2000 J. Biotechnol. 76(2-3): 245-251). Various yeasts, such as K.lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha,have played particularly important roles as eukaryotic expressionsystems because they are able to grow to high cell densities and secretelarge quantities of recombinant protein. Likewise, filamentous fungi,such as Aspergillus niger, Fusarium sp, Neurospora crassa and others,have been used to efficiently produce glycoproteins at the industrialscale. However, as noted above, glycoproteins expressed in any of theseeukaryotic microorganisms differ substantially in N-glycan structurefrom those in animals. This has prevented the use of yeast orfilamentous fungi as hosts for the production of many therapeuticglycoproteins.

Although glycosylation in yeast and fungi is very different than inhumans, some common elements are shared. The first step, the transfer ofthe core oligosaccharide structure to the nascent protein, is highlyconserved in all eukaryotes including yeast, fungi, plants and humans(compare FIGS. 1A and 1B). Subsequent processing of the coreoligosaccharide, however, differs significantly in yeast and involvesthe addition of several mannose sugars. This step is catalyzed bymannosyltransferases residing in the Golgi (e.g. OCH1, MNT1, MNN1,etc.), which sequentially add mannose sugars to the coreoligosaccharide. The resulting structure is undesirable for theproduction of humanoid proteins and it is thus desirable to reduce oreliminate mannosyltransferase activity. Mutants of S. cerevisiaedeficient in mannosyltransferase activity (e.g. och1 or mnn9 mutants)have shown to be non-lethal and display a reduced mannose content in theoligosaccharide of yeast glycoproteins. Other oligosaccharide processingenzymes, such as mannosylphosphate transferase, may also have to beeliminated depending on the host's particular endogenous glycosylationpattern. After reducing undesired endogenous glycosylation reactions,the formation of complex N-glycans has to be engineered into the hostsystem. This requires the stable expression of several enzymes andsugar-nucleotide transporters. Moreover, one has to localize theseenzymes so that a sequential processing of the maturing glycosylationstructure is ensured.

Several efforts have been made to modify the glycosylation pathways ofeukaryotic microorganisms to provide glycoproteins more suitable for useas mammalian therapeutic agents. For example, severalglycosyltransferases have been separately cloned and expressed in S.cerevisiae (GalT, GnTI), Aspergillus nidulans (GnTI) and other fungi(Yoshida et al., 1999, Kalsner et al., 1995 Glycoconj. J. 12(3):360-370,Schwientek et al., 1995). However, N-glycans resembling those made inhuman cells were not obtained.

Yeasts produce a variety of mannosyltransferases (e.g.,1,3-mannosyltransferases such as MNN1 in S. cerevisiae; Graham and Emr,1991 J. Cell. Biol. 114(2):207-218), 1,2-mannosyltransferases (e.g.KTR/KRE family from S. cerevisiae), 1,6-mannosyltransferases (e.g., OCH1from S. cerevisiae), mannosylphosphate transferases and their regulators(e.g., MNN4 and MNN6 from S. cerevisiae) and additional enzymes that areinvolved in endogenous glycosylation reactions. Many of these genes havebeen deleted individually giving rise to viable organisms having alteredglycosylation profiles. Examples are shown in Table 1.

TABLE 1 Examples of yeast strains having altered mannosylation StrainN-glycan (wild type) Mutation N-glycan (mutant) Reference S. pombeMan_(>9)GlcNAc₂ OCH1 Man₈GlcNAc₂ Yoko-o et al. , 2001 FEBS Lett. 489(1):75-80 S. cerevisiae Man_(>9)GlcNAc₂ OCH1/ Man₈GlcNAc₂ Nakanishi-ShindoMNN1 et al, . 1993 J. Biol. Chem. 268(35): 26338-26345 S. cerevisiaeMan_(>9)GlcNAc₂ OCH1/ Man₈GlcNAc₂ Chiba et al. , 1998 MNN1/ J. Biol.Chem. MNN4 273, 26298-26304 P. pastoris Hyperglycosylated OCH1 NotWelfide, Japanese (complete hyperglycosylated Application deletion)Publication No. 8- 336387 P. pastoris Man_(>8)GlcNAc₂ OCH1Man_(>8)GlcNAc₂ Contreras et al. (disruption) WO 02/00856 A2

Japanese Patent Application Publication No. 8-336387 discloses thedeletion of an OCH1 homolog in Pichia pastoris. In S. cerevisiae, OCH1encodes a 1,6-mannosyltransferase, which adds a mannose to the glycanstructure Man₈GlcNAc₂ to yield Man₉GlcNAc₂. The Man₉GlcNAc₂ structure,which contains three 1,6 mannose residues, is then a substrate forfurther 1,2-, 1,6-, and 1,3-mannosyltransferases in vivo, leading to thehypermannosylated glycoproteins that are characteristic for S.cerevisiae and which typically may have 30-40 mannose residues perN-glycan. Because the Och1p initiates the transfer of 1,6 mannose to theMan₈GlcNAc₂ core, it is often referred to as the “initiating 1,6mannosyltransferase” to distinguish it from other 1,6mannosyltransferases acting later in the Golgi. In an och1 mnn1 mnn4mutant strain of S. cerevisiae, proteins glycosylated with Man₈GlcNAc₂accumulate and hypermannosylation does not occur. However, Man₈GlcNAc₂is not a substrate for mammalian glycosyltransferases, such as humanUDP-GlcNAc transferase I, and accordingly, the use of that mutantstrain, in itself, is not useful for producing mammalian-like proteins,i.e., with complex or hybrid glycosylation patterns.

Although Japanese Patent Application Publication No. 8-336387 disclosesmethods to obtain an och1 mutant of P. pastoris displaying a reducedmannosylation phenotype, it provides no data on whether the initiating1,6 mannosyltransferase activity presumed to be encoded by OCH1 isreduced or eliminated. It is well-established in the field of fungalgenetics that homologs of genes often do not play the same role in theirrespective host organism. For example, the Neurospora rca-1 genecomplements an Aspergillus flbD sporulation mutant but has noidentifiable role in Neurospora sporulation. Shen, W. C. et al.,Genetics 1998; 148(3):1031-41. More recently, Contreras (WO 02/00856 A2)shows that, in an och1 mutant of P. pastoris, at least 50% of the cellwall glycans cannot be trimmed to Man₅GlcNAc₂ with a Trichoderma reeseiα-1,2-mannosidase (see FIG. 11 of WO 02/00856 A2). As the wild-typedisplays a very similar glycosylation pattern (FIG. 10, Panel 2 of WO02/00856 A2), it appears that the OCH1 gene of P. pastoris may notencode the initiating 1,6-mannosyltransferase activity and is thusdifferent from its genetic homolog in S. cerevisiae. Thus, to date,there is no evidence that initiating α-1,6-mannosyltransferase activityis eliminated in och1 mutants of P. pastoris, which further supports thenotion that the glycosylation pathways of S. cerevisiae and P. pastorisare significantly different.

Martinet et al. (Biotechnol. Lett. 1998, 20(12), 1171-1177) reported theexpression of α-1,2-mannosidase from T reesei in P. pastoris. Somemannose trimming from the N-glycans of a model protein was observed.However, the model protein had no N-glycans with the structureMan₅GlcNAc₂, which would be necessary as an intermediate for thegeneration of complex N-glycans. Accordingly, that system is not usefulfor producing proteins with complex or hybrid glycosylation patterns.

Similarly, Chiba et al. (1998) expressed α-1,2-mannosidase fromAspergillus saitoi in the yeast Saccharomyces cerevisiae. A signalpeptide sequence (His-Asp-Glu-Leu) (SEQ ID NO:5) was engineered into theexogenous mannosidase to promote its retention in the endoplasmicreticulum. In addition, the yeast host was a mutant lacking enzymeactivities associated with hypermannosylation of proteins:1,6-mannosyltransferase (och1); 1,3-mannosyltransferase (mnn1); and aregulator of mannosylphosphate transferase (mnn4). The N-glycans of thetriple mutant host consisted primarily of the structure Man₈GlcNAc₂,rather than the high mannose forms found in wild-type S. cerevisiae. Inthe presence of the engineered mannosidase, the N-glycans of a modelprotein (carboxypeptidase Y) were trimmed to give a mixture consistingof 27 mole % Man₅GlcNAc₂, 22 mole % Man₆GlcNAc₂, 22 mole % Man₇GlcNAc₂,and 29 mole % Man₈GlcNAc₂. Trimming of cell wall glycoproteins was lessefficient, with only 10 mole % of the N-glycans having the desiredMan₅GlcNAc₂ structure.

Even if all the Man₅GlcNAc₂ glycans were the correct Man₅GlcNAc₂ formthat can be converted to GlcNAcMan₅GlcNAc₂ by GnTI, the above systemwould not be efficient for the production of proteins having human-likeglycosylation patterns. If several glycosylation sites are present in adesired protein, the probability (P) of obtaining such a protein in acorrect form follows the relationship P=(F)^(n), where n equals thenumber of glycosylation sites, and F equals the fraction of desiredglycoforms. A glycoprotein with three glycosylation sites would have a0.1% chance of providing the appropriate precursors for complex andhybrid N-glycan processing on all of its glycosylation sites. Thus,using the system of Chiba to make a glycoprotein having a singleN-glycosylation site, at least 73 mole % would have an incorrectstructure. For a glycoprotein having two or three N-glycosylation sites,at least 93 or 98 mole % would have an incorrect structure,respectively. Such low efficiencies of conversion are unsatisfactory forthe production of therapeutic agents, particularly as the separation ofproteins having different glycoforms is typically costly and difficult.

Chiba et al. (WO 01/14522) have shown high levels of Man₅GlcNAc₂structures on recombinant fibroblast growth factor (FGF), a secretedsoluble glycoprotein produced in S. cerevisiae. It is not clear,however, that the detected Man₅GlcNAc₂ was produced inside the host cell(i.e. in vivo) because the α-1,2 mannosidase was targeted by fusion withan HDEL (SEQ ID NO:5) localization tag, a mechanism, which is known tobe leaky (Pelham H. R. (1998) EMBO J. 7, 913-918). It is more likelythat FGF was secreted into the medium, where it was then processed byα-1,2 mannosidase which had escaped the HDEL (SEQ ID NO:5) retrievalmechanism and leaked into the medium. As mentioned above, anintracellular protein (CPY), expressed in the same strain, containedmostly glycans (more than 73%) that were Man₆GlcNAc₂ and larger. Themajority of the Man₅GlcNAc₂ structures on FGF are, thus, likely to havebeen produced ex vivo. It is further unclear whether the Man₅GlcNAc₂structures that were produced were productive substrates for GnTI.

As the above work demonstrates, one can trim Man₈GlcNAc₂ structures to aMan₅GlcNAc₂ isomer in S. cerevisiae, although high efficiency trimminggreater than 50% in vivo has yet to be determined, by engineering afungal mannosidase from A. saitoi into the endoplasmic reticulum (ER).The shortcomings of this approach are two-fold: (1) it is not clearwhether the Man₅GlcNAc₂ structures formed are in fact formed in vivo(rather than having been secreted and further modified by mannosidasesoutside the cell); and (2) it is not clear whether any Man₅GlcNAc₂structures formed, if in fact formed in vivo, are the correct isoform tobe a productive substrate for subsequent N-glycan modification by GlcNActransferase I (Maras et al., 1997, Eur. J. Biochem. 249, 701-707).

With the objective of providing a more human-like glycoprotein derivedfrom a fungal host, U.S. Pat. No. 5,834,251 discloses a method forproducing a hybrid glycoprotein derived from Trichoderma reseei. Ahybrid N-glycan has only mannose residues on the Manα1-6 arm of the coremannose structure and one or two complex antennae on the Manα1-3 arm.While this structure has utility, the method has the disadvantage thatnumerous enzymatic steps must be performed in vitro, which is costly andtime-consuming. Isolated enzymes are expensive to prepare and needcostly substrates (e.g. UDP-GlcNAc). The method also does not allow forthe production of complex glycans on a desired protein.

Intracellular Mannosidase Activity Involved in N-Glycan Trimming

Alpha-1,2-mannosidase activity is required for the trimming ofMan₈GlcNAc₂ to form Man₅GlcNAc₂, which is a major intermediate forcomplex N-glycan formation in mammals. Previous work has shown thattruncated murine, fungal and human α-1,2-mannosidase can be expressed inthe methylotropic yeast P. pastoris and display Man₈GlcNAc₂ toMan₅GlcNAc₂ trimming activity (Lal et al., Glycobiology 1998 October;8(10):981-95; Tremblay et al., Glycobiology 1998 June; 8(6):585-95,Callewaert et al., 2001). However, to date, no reports exist that showthe high level in vivo trimming of Man₈GlcNAc₂ to Man₅GlcNAc₂ on asecreted glycoprotein from P. pastoris.

While it is useful to engineer strains that are able to produceMan₅GlcNAc₂ as the primary N-glycan structure, any attempt to furthermodify these high mannose precursor structures to more closely resemblehuman glycans requires additional in vivo or in vitro steps. Methods tofurther humanize glycans from fungal and yeast sources in vitro aredescribed in U.S. Pat. No. 5,834,251 (supra). As discussed above,however, if Man₅GlcNAc₂ is to be further humanized in vivo, one has toensure that the generated Man₅GlcNAc₂ structures are, in fact, generatedintracellularly and not the product of mannosidase activity in themedium. Complex N-glycan formation in yeast or fungi will require highlevels of Man₅GlcNAc₂ to be generated within the cell because onlyintracellular Man₅GlcNAc₂ glycans can be further processed to hybrid andcomplex N-glycans in vivo. In addition, one has to demonstrate that themajority of Man₅GlcNAc₂ structures generated are in fact a substrate forGnTI and thus allow the formation of hybrid and complex N-glycans.

Moreover, the mere presence of an α-1,2-mannosidase in the cell doesnot, by itself, ensure proper intracellular trimming of Man₈GlcNAc₂ toMan₅GlcNAc₂. (See, e.g., Contreras et al. WO 02/00856 A2, in which anHDEL (SEQ ID NO:5) tagged mannosidase of T. reesei is localizedprimarily in the ER and co-expressed with an influenza haemagglutinin(HA) reporter protein on which virtually no Man₅GlcNAc₂ could bedetected. See also Chiba et al., 1998 (supra), in which a chimericα-1,2-mannosidase/Och1p transmembrane domain fusion localized in the ER,early Golgi and cytosol of S. cerevisiae, had no mannosidase trimmingactivity). Accordingly, mere localization of a mannosidase in the ER orGolgi is insufficient to ensure activity of the respective enzyme inthat targeted organelle. (See also, Martinet et al. (1998), supra,showing that α-1,2-mannosidase from T. reesei, while localizingintracellularly, increased rather than decreased the extent ofmannosylation). To date, there is no report that demonstrates theintracellular localization of an active heterologous α-1,2-mannosidasein either yeast or fungi using a transmembrane localization sequence.

Accordingly, the need exists for methods to produce glycoproteinscharacterized by a high intracellular Man₅GlcNAc₂ content which can befurther processed into human-like glycoprotein structures in non-humaneukaryotic host cells, and particularly in yeast and filamentous fungi.

SUMMARY OF THE INVENTION

Host cells and cell lines having genetically modified glycosylationpathways that allow them to carry out a sequence of enzymatic reactionswhich mimic the processing of glycoproteins in mammals, especially inhumans, have been developed. Recombinant proteins expressed in theseengineered hosts yield glycoproteins more similar, if not substantiallyidentical, to their mammalian, e.g., human counterparts. Host cells ofthe invention, e.g., lower eukaryotic microorganisms and othernon-human, eukaryotic host cells grown in culture, are modified toproduce N-glycans such as Man₅GlcNAc₂ or other structures produced alonghuman glycosylation pathways. This is achieved using a combination ofengineering and/or selection of strains which: do not express certainenzymes which create the undesirable structures characteristic of thefungal glycoproteins; which express heterologous enzymes selected eitherto have optimal activity under the conditions present in the host cellwhere activity is to be achieved; or combinations thereof; wherein thegenetically engineered eukaryote expresses at least one heterologousenzyme activity required to produce a “human-like” glycoprotein. Hostcells of the invention may be modified further by heterologousexpression of one or more activities such as glycosyltransferases, sugartransporters and mannosidases, to become strains for the production ofmammalian, e.g., human therapeutic glycoproteins.

The present invention thus provides a glycoprotein production methodusing (1) a lower eukaryotic host such as a unicellular or filamentousfungus, or (2) any non-human eukaryotic organism that has a differentglycosylation pattern from humans, to modify the glycosylationcomposition and structures of the proteins made in a host organism(“host cell”) so that they resemble more closely carbohydrate structuresfound in mammalian, e.g., human proteins. The process allows one toobtain an engineered host cell which can be used to express and targetany desirable gene(s), e.g., one involved in glycosylation, by methodsthat are well-established in the scientific literature and generallyknown to the artisan in the field of protein expression. Host cells withmodified oligosaccharides are created or selected. N-glycans made in theengineered host cells have a Man₅GlcNAc₂ core structure which may thenbe modified further by heterologous expression of one or more enzymes,e.g., glycosyltransferases, glycosidases, sugar transporters andmannosidases, to yield human-like glycoproteins. For the production oftherapeutic proteins, this method may be adapted to engineer cell linesin which any desired glycosylation structure may be obtained.

Accordingly, in one embodiment, the invention provides a method forproducing a human-like glycoprotein in a non-human eukaryotic host cell.The host cell of the invention is selected or engineered to be depletedin 1,6-mannosyltransferase activities which would otherwise add mannoseresidues onto the N-glycan on a glycoprotein. One or more enzymes(enzymatic activities) are introduced into the host cell which enablethe production of a Man₅GlcNAc₂ carbohydrate structure at a high yield,e.g., at least 30 mole percent. In a more preferred embodiment, at least10% of the Man₅GlcNAc₂ produced within the host cell is a productivesubstrate for GnTI and thus for further glycosylation reactions in vivoand/or in vitro that produce a finished N-glycan that is similar oridentical to that formed in mammals, especially humans.

In another embodiment, a nucleic acid molecule encoding one or moreenzymes for production of a Man₅GlcNAc₂ carbohydrate structure isintroduced into a host cell selected or engineered to be depleted in1,6-mannosyltransferase activities. In one preferred embodiment, atleast one enzyme introduced into the host cell is selected to haveoptimal activity at the pH of the subcellular location where thecarbohydrate structure is produced. In another preferred embodiment, atleast one enzyme is targeted to a host subcellular organelle where theenzyme will have optimal activity, e.g., by means of a chimeric proteincomprising a cellular targeting signal peptide not normally associatedwith the enzyme.

The invention further provides isolated nucleic acid molecules andvectors comprising such molecules which encode an initiatingα1,6-mannosyltransferase activity isolated from P. pastoris or from K.lactis. These nucleic acid molecules comprise sequences that arehomologous to the OCH1 gene in S. cerevisiae. These and homologoussequences are useful for constructing host cells which will nothypermannosylate the N-glycan of a glycoprotein.

In another embodiment, the host cell is engineered to express aheterologous glycosidase, e.g., by introducing into the host one or morenucleic acid molecules encoding the glycosidase. Preferably, a nucleicacid molecule encodes one or more mannosidase activities involved in theproduction of Man₅GlcNAc₂ from Man₈GlcNAc₂ or Man₉GlcNAc₂. In apreferred embodiment, at least one of the encoded mannosidase activitieshas a pH optimum within 1.4 pH units of the average pH optimum of otherrepresentative enzymes in the organelle in which the mannosidaseactivity is localized, or has optimal activity at a pH of between about5.1 and about 8.0, preferably between about 5.5 and about 7.5.Preferably, the heterologous enzyme is targeted to the endoplasmicreticulum, the Golgi apparatus or the transport vesicles between ER andGolgi of the host organism, where it trims N-glycans such as Man₈GlcNAc₂to yield high levels of Man₅GlcNAc₂. In one embodiment, the enzyme istargeted by forming a fusion protein between a catalytic domain of theenzyme and a cellular targeting signal peptide, e.g., by the in-frameligation of a DNA fragment encoding a cellular targeting signal peptidewith a DNA fragment encoding a glycosylation enzyme or catalyticallyactive fragment thereof.

In yet another embodiment, the glycosylation pathway of a host ismodified to express a sugar nucleotide transporter. In a preferredembodiment, a nucleotide diphosphatase enzyme is also expressed. Thetransporter and diphosphatase improve the efficiency of engineeredglycosylation steps, by providing the appropriate substrates for theglycosylation enzymes in the appropriate compartments, reducingcompetitive product inhibition, and promoting the removal of nucleosidediphosphates.

The present invention also provides a combinatorial nucleic acid libraryuseful for making fusion constructs which can target a desired proteinor polypeptide fragment, e.g., an enzyme involved in glycosylation or acatalytic domain thereof, to a selected subcellular region of a hostcell. In one preferred embodiment, the combinatorial nucleic acidlibrary comprises (a) nucleic acid sequences encoding different cellulartargeting signal peptides and (b) nucleic acid sequences encodingdifferent polypeptides to be targeted. Nucleic acid sequences of orderived from (a) and (b) are ligated together to produce fusionconstructs, at least one of which encodes a functional protein domain(e.g., a catalytic domain of an enzyme) ligated in-frame to aheterologous cellular targeting signal peptide, i.e., one which itnormally does not associate with.

The invention also provides a method for modifying the glycosylationpathway of a host cell (e.g., any eukaryotic host cell, including ahuman host cell) using enzymes involved in modifying N-glycans includingglycosidases and glycosyltransferases; by transforming the host cellwith a nucleic acid (e.g., a combinatorial) library of the invention toproduce a genetically mixed cell population expressing at least one andpreferably two or more distinct chimeric glycosylation enzymes having acatalytic domain ligated in-frame to a cellular targeting signal peptidewhich it normally does not associate with. A host cell having a desiredglycosylation phenotype may optionally be selected from the population.Host cells modified using the library and associated methods of theinvention are useful, e.g., for producing glycoproteins having aglycosylation pattern similar or identical to those produced in mammals,especially humans.

In another aspect, the combinatorial library of the present inventionenables production of one or a combination of catalytically activeglycosylation enzymes, which successfully localize to intracellularcompartments in which they function efficiently in theglycosylation/secretory pathway. Preferred enzymes convertMan₅(α-1,2-Man)₃₋₉GlcNAc₂ to Man₅GlcNAc₂ at high efficiency in vivo. Inaddition, the invention provides eukaryotic host strains, and inparticular, yeasts, fungal, insect, plant, plant cells, algae and insectcell hosts, capable of producing glycoprotein intermediates or productswith Man₅GlcNAc₂ and/or GlcNAcMan₅GlcNAc₂ as the predominant N-glycan.

The present invention also provides recombinant molecules derived from acombinatorial nucleic acid library; vectors, including expressionvectors, comprising such recombinant molecules; proteins encoded by therecombinant molecules and vectors; host cells transformed with therecombinant molecules or vectors; glycoproteins produced from suchtransformed hosts; and methods for producing, in vivo, glycoproteinintermediates or products with predominantly Man₅GlcNAc₂ orGlcNAcMan₅GlcNAc₂ N-glycans covalently attached to appropriateglycosylation sites using the combinatorial library.

Further aspects of this invention include methods, compositions and kitsfor diagnostic and therapeutic uses in which presence or absence ofMan₅GlcNAc₂ and/or GlcNAcMan₅GlcNAc₂ on a glycoprotein may be detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram of a typical fungal N-glycosylationpathway.

FIG. 1B is a schematic diagram of a typical human N-glycosylationpathway.

FIG. 2 depicts construction of a combinatorial DNA library of fusionconstructs. FIG. 2A diagrams the insertion of a targeting peptidefragment into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.). FIG. 2B showsthe generated targeting peptide sub-library having restriction sitesNotI-AsaI. FIG. 2C diagrams the insertion of a catalytic domain regioninto pJN347, a modified pUC19 vector. FIG. 2D shows the generatedcatalytic domain sub-library having restriction sites NotI, AscI andPacI. FIG. 2E depicts one particular fusion construct generated from thetargeting peptide sub-library and the catalytic domain sub-library.

FIG. 3 (SEQ ID NOS 45-46 respectively, in order of appearance)illustrates the M. musculus α-1,2-mannosidase IA open reading frame. Thesequences of the PCR primers used to generate N-terminal truncations areunderlined.

FIG. 4 illustrates engineering of vectors with multiple auxotrophicmarkers and genetic integration of target proteins in the P. pastorisOCH1 locus.

FIG. 5 shows MALDI-TOF analysis demonstrating production of kringle 3domain of human plasminogen (K3) glycoproteins having Man₅GlcNAc₂ as thepredominant N-glycan structure in P. pastoris. FIG. 5A depicts thestandard Man₅GlcNAc₂ [a] glycan (Glyko, Novato, Calif.) andMan₅GlcNAc₂+Na⁺ [b]. FIG. 5B shows PNGase-released glycans from K3 wildtype. The N-glycans shown are as follows: Man₉GlcNAc₂ [d]; Man₁₀GlcNAc₂[e]; Man₁₁GlcNAc_(2 [)1]; Man₁₂GlcNAc₂ [g]. FIG. 5C depicts the och1deletion resulting in the production of Man₈GlcNAc₂ [c] as thepredominant N-glycan. FIGS. 5D and 5E show the production of Man₅GlcNAc₂[b] after in vivo trimming of Man₈GlcNAc₂ with a chimericα-1,2-mannosidase. The predominant N-glycan is indicated by a peak witha mass (m/z) of 1253 consistent with its identification as Man₅GlcNAc₂[b].

FIG. 6 shows MALDI-TOF analysis demonstrating production of IFN-βglycoproteins having Man₅GlcNAc₂ as the predominant N-glycan structurein P. pastoris. FIG. 6A shows the standard Man₅GlcNAc₂ [a] andMan₅GlcNAc₂+Na⁺ [b] as the standard (Glyko, Novato, Calif.). FIG. 6Bshows PNGase-released glycans from IFN-β wildtype. FIG. 6C depicts theoch1 knockout producing Man₈GlcNAc₂ [c]; Man₉GlcNAc₂ [d]; Man₁₀GlcNAc₂[e]; Man₁₁GlcNAc_(2 [f]); Man₁₂GlcNAc₂ [g]; and no production ofMan₅GlcNAc₂ [b]. FIG. 6D shows relatively small amount of Man₅GlcNAc₂[b] among other intermediate N-glycans Man₈GlcNAc₂ [c] to Man₁₂GlcNAc₂[g]. FIG. 6E shows a significant amount of Man₅GlcNAc₂ [b] relative tothe other glycans Man₈GlcNAc₂ [c] and Man₉GlcNAc₂ [d] produced by pGC5(Saccharomyces MNS1(m)/mouse mannosidase IB Δ99). FIG. 6F showspredominant production of Man₅GlcNAc₂ [b] on the secreted glycoproteinIFN-β by pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187). TheN-glycan is indicated by a peak with a mass (m/z) of 1254 consistentwith its identification as Man₅GlcNAc₂ [b].

FIG. 7 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂standard labeled with 2-AB (negative control); (B) supernatant of mediumP pastoris, Δoch1 transformed with pFB8 mannosidase, which demonstratesa lack of extracellular mannosidase activity in the supernatant; and (C)Man₉GlcNAc₂ standard labeled with 2-AB after exposure to T. reeseimannosidase (positive control).

FIG. 8 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂standard labeled with 2-AB (negative control); (B) supernatant of mediumP. pastoris, Δoch1 transformed with pGC5 mannosidase, which demonstratesa lack of extracellular mannosidase activity in the supernatant; and (C)Man₉GlcNAc₂ standard labeled with 2-AB after exposure to T. reeseimannosidase (positive control).

FIG. 9 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂standard labeled with 2-AB (negative control); (B) supernatant of mediumP. pastoris, Δoch1 transformed with pBC18-5 mannosidase, whichdemonstrates lack of extracellular mannosidase activity in thesupernatant; and (C) supernatant of medium P. pastoris, Δoch1transformed with pDD28-3, which demonstrates activity in the supernatant(positive control).

FIG. 10 demonstrates the activity of an UDP-GlcNAc transporter in theproduction of GlcNAcMan₅GlcNAc₂ in P. pastoris. FIG. 10A depicts a P.pastoris strain (YSH-3) with a human GnTI but without the UDP-GlcNActransporter resulting in some production of GlcNAcMan₅GlcNAc₂ [b] but apredominant production of Man₅GlcNAc₂ [a]. FIG. 10B depicts the additionof UDP-GlcNAc transporter from K. lactis in a strain (PBP-3) with thehuman GnTI, which resulted in the predominant production ofGlcNAcMan₅GlcNAc₂ [b]. The single prominent peak of mass (m/z) at 1457is consistent with its identification as GlcNAcMan₅GlcNAc₂ [b] as shownin FIG. 10B.

FIG. 11 shows a pH optimum of a heterologous mannosidase enzyme encodedby pBB27-2 (Saccharomyces MNN10 (s)/C. elegans mannosidase IB Δ31)expressed in P. pastoris.

FIG. 12 shows MALDI-TOF analysis of N-glycans released from a cell freeextract of K. lactis. FIG. 12A shows the N-glycans released fromwild-type cells, which includes high-mannose type N-glycans. FIG. 12Bshows the N-glycans released from och1 mnn1 deleted cells, revealing adistinct peak of mass (m/z) at 1908 consistent with its identificationas Man₉GlcNAc₂ [d]. FIG. 12C shows the N-glycans released from och1 mnn1deleted cells after in vitro α-1,2-mannosidase digest corresponding to apeak consistent with Man₅GlcNAc₂.

FIG. 13 represents T-DNA cassettes with catalytic domain(s) ofglycosylation enzymes fused in-frame to different leader sequences. Theends of the T-DNA are marked by the right (rb) and left borders (lb).Various promoters and terminators may also be used. The plant selectablemarker can also be varied. The right and left borders are required onlyfor agrobacterium-mediated transformation and not for particlebombardment or electroporation.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present invention are generally performed according toconventional methods well known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well-known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Harlow andLane Antibodies: A Laboratory Manual Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Introduction to Glycobiology,Maureen E. Taylor, Kurt Drickamer, Oxford Univ. Press (2003);Worthington Enzyme Manual, Worthington Biochemical Corp. Freehold, N.J.;Handbook of Biochemistry: Section A Proteins Vol 11976 CRC Press;Handbook of Biochemistry: Section A Proteins Vol II 1976 CRC Press;Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).The nomenclatures used in connection with, and the laboratory proceduresand techniques of, molecular and cellular biology, protein biochemistry,enzymology and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art.

All publications, patents and other references mentioned herein areincorporated by reference.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the term “N-glycan” refers to an N-linkedoligosaccharide, e.g., one that is attached by anasparagine-N-acetylglucosamine linkage to an asparagine residue of apolypeptide. N-glycans have a common pentasaccharide core of Man₃GlcNAc₂(“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). The term “trimannosecore” used with respect to the N-glycan also refers to the structureMan₃GlcNAc₂ (“Man₃”). N-glycans differ with respect to the number ofbranches (antennae) comprising peripheral sugars (e.g., fucose andsialic acid) that are added to the Man₃ core structure. N-glycans areclassified according to their branched constituents (e.g., high mannose,complex or hybrid).

A “high mannose” type N-glycan has five or more mannose residues. A“complex” type N-glycan typically has at least one GlcNAc attached tothe 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannosearm of the trimannose core. Complex N-glycans may also have galactose(“Gal”) residues that are optionally modified with sialic acid orderivatives (“NeuAc”, where “Neu” refers to neuraminic acid and “Ac”refers to acetyl). A complex N-glycan typically has at least one branchthat terminates in an oligosaccharide such as, for example: NeuNAc-;NeuAca2-6GalNAca1-; NeuAca2-3Galb1-3GalNAca1-;NeuAca2-3/6Galb1-4GlcNAcb1-; GlcNAca1-4Galb1-(mucins only);Fuca1-2Galb1-(blood group H). Sulfate esters can occur on galactose,GalNAc, and GlcNAc residues, and phosphate esters can occur on mannoseresidues. NeuAc (Neu: neuraminic acid; Ac:acetyl) can be O-acetylated orreplaced by NeuGl (N-glycolylneuraminic acid). Complex N-glycans mayalso have intrachain substitutions comprising “bisecting” GlcNAc andcore fucose (“Fuc”). A “hybrid” N-glycan has at least one GlcNAc on theterminal of the 1,3 mannose arm of the trimannose core and zero or moremannoses on the 1,6 mannose arm of the trimannose core.

The term “predominant” or “predominantly” used with respect to theproduction of N-glycans refers to a structure which represents the majorpeak detected by matrix assisted laser desorption ionization time offlight mass spectrometry (MALDI-TOF) analysis.

Abbreviations used herein are of common usage in the art, see, e.g.,abbreviations of sugars, above. Other common abbreviations include“PNGase”, which refers to peptide N-glycosidase F (EC 3.2.2.18); “GlcNAcTr” or “GnT,” which refers to N-acetylglucosaminyl Transferase enzymes;“NANA” refers to N-acetylneuraminic acid.

As used herein, a “humanized glycoprotein” or a “human-likeglycoprotein” refers alternatively to a protein having attached theretoN-glycans having less than four mannose residues, and syntheticglycoprotein intermediates (which are also useful and can be manipulatedfurther in vitro or in vivo) having at least five mannose residues.Preferably, glycoproteins produced according to the invention contain atleast 30 mole %, preferably at least 40 mole % and more preferably50-100 mole % of the Man₅GlcNAc₂ intermediate, at least transiently.This may be achieved, e.g., by engineering a host cell of the inventionto express a “better”, i.e., a more efficient glycosylation enzyme. Forexample, a mannosidase is selected such that it will have optimalactivity under the conditions present at the site in the host cell whereproteins are glycosylated and is introduced into the host cellpreferably by targeting the enzyme to a host cell organelle whereactivity is desired.

The term “enzyme”, when used herein in connection with altering hostcell glycosylation, refers to a molecule having at least one enzymaticactivity, and includes full-length enzymes, catalytically activefragments, chimerics, complexes, and the like. A “catalytically activefragment” of an enzyme refers to a polypeptide having a detectable levelof functional (enzymatic) activity.

A lower eukaryotic host cell, when used herein in connection withglycosylation profiles, refers to any eukaryotic cell which ordinarilyproduces high mannose containing N-glycans, and thus is meant to includesome animal or plant cells and most typical lower eukaryotic cells,including uni- and multicellular fungal and algal cells.

As used herein, the term “secretion pathway” refers to the assembly lineof various glycosylation enzymes to which a lipid-linked oligosaccharideprecursor and an N-glycan substrate are sequentially exposed, followingthe molecular flow of a nascent polypeptide chain from the cytoplasm tothe endoplasmic reticulum (ER) and the compartments of the Golgiapparatus. Enzymes are said to be localized along this pathway. Anenzyme X that acts on a lipid-linked glycan or an N-glycan before enzymeY is said to be or to act “upstream” to enzyme Y; similarly, enzyme Y isor acts “downstream” from enzyme X.

The term “targeting peptide” as used herein refers to nucleotide oramino acid sequences encoding a cellular targeting signal peptide whichmediates the localization (or retention) of an associated sequence tosub-cellular locations, e.g., organelles.

The term “polynucleotide” or “nucleic acid molecule” refers to apolymeric form of nucleotides of at least 10 bases in length. The termincludes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNAmolecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA orRNA containing non-natural nucleotide analogs, non-nativeinternucleoside bonds, or both. The nucleic acid can be in anytopological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hairpinned, circular, or in apadlocked conformation. The term includes single and double strandedforms of DNA. A nucleic acid molecule of this invention may include bothsense and antisense strands of RNA, cDNA, genomic DNA, and syntheticforms and mixed polymers of the above. They may be modified chemicallyor biochemically or may contain non-natural or derivatized nucleotidebases, as will be readily appreciated by those of skill in the art. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),pendent moieties (e.g., polypeptides), intercalators (e.g., acridine,psoralen, etc.), chelators, alkylators, and modified linkages (e.g.,alpha anomeric nucleic acids, etc.) Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known in the art and include, forexample, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule.

Unless otherwise indicated, a “nucleic acid comprising SEQ ID NO:X”refers to a nucleic acid, at least a portion of which has either (i) thesequence of SEQ ID NO:X, or (ii) a sequence complementary to SEQ IDNO:X. The choice between the two is dictated by the context. Forinstance, if the nucleic acid is used as a probe, the choice between thetwo is dictated by the requirement that the probe be complementary tothe desired target.

An “isolated” or “substantially pure” nucleic acid or polynucleotide(e.g., an RNA, DNA or a mixed polymer) is one which is substantiallyseparated from other cellular components that naturally accompany thenative polynucleotide in its natural host cell, e.g., ribosomes,polymerases, and genomic sequences with which it is naturallyassociated. The term embraces a nucleic acid or polynucleotide that (1)has been removed from its naturally occurring environment, (2) is notassociated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (3) is operatively linkedto a polynucleotide which it is not linked to in nature, or (4) does notoccur in nature. The term “isolated” or “substantially pure” also can beused in reference to recombinant or cloned DNA isolates, chemicallysynthesized polynucleotide analogs, or polynucleotide analogs that arebiologically synthesized by heterologous systems.

However, “isolated” does not necessarily require that the nucleic acidor polynucleotide so described has itself been physically removed fromits native environment. For instance, an endogenous nucleic acidsequence in the genome of an organism is deemed “isolated” herein if aheterologous sequence (i.e., a sequence that is not naturally adjacentto this endogenous nucleic acid sequence) is placed adjacent to theendogenous nucleic acid sequence, such that the expression of thisendogenous nucleic acid sequence is altered. By way of example, anon-native promoter sequence can be substituted (e.g., by homologousrecombination) for the native promoter of a gene in the genome of ahuman cell, such that this gene has an altered expression pattern. Thisgene would now become “isolated” because it is separated from at leastsome of the sequences that naturally flank it.

A nucleic acid is also considered “isolated” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “isolated” if it contains an insertion, deletion or a pointmutation introduced artificially, e.g., by human intervention. An“isolated nucleic acid” also includes a nucleic acid integrated into ahost cell chromosome at a heterologous site, a nucleic acid constructpresent as an episome. Moreover, an “isolated nucleic acid” can besubstantially free of other cellular material, or substantially free ofculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

As used herein, the phrase “degenerate variant” of a reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the standard genetic code, to provide an amino acidsequence identical to that translated from the reference nucleic acidsequence.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over a stretch of at least aboutnine nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides. There are a number of different algorithmsknown in the art which can be used to measure nucleotide sequenceidentity. For instance, polynucleotide sequences can be compared usingFASTA, Gap or Bestfit, which are programs in Wisconsin Package Version10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences (Pearson, 1990, hereinincorporated by reference). For instance, percent sequence identitybetween nucleic acid sequences can be determined using FASTA with itsdefault parameters (a word size of 6 and the NOPAM factor for thescoring matrix) or using Gap with its default parameters as provided inGCG Version 6.1, herein incorporated by reference.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 50%, more preferably 60%of the nucleotide bases, usually at least about 70%, more usually atleast about 80%, preferably at least about 90%, and more preferably atleast about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap, as discussed above.

Alternatively, substantial homology or similarity exists when a nucleicacid or fragment thereof hybridizes to another nucleic acid, to a strandof another nucleic acid, or to the complementary strand thereof, understringent hybridization conditions. “Stringent hybridization conditions”and “stringent wash conditions” in the context of nucleic acidhybridization experiments depend upon a number of different physicalparameters. Nucleic acid hybridization will be affected by suchconditions as salt concentration, temperature, solvents, the basecomposition of the hybridizing species, length of the complementaryregions, and the number of nucleotide base mismatches between thehybridizing nucleic acids, as will be readily appreciated by thoseskilled in the art. One having ordinary skill in the art knows how tovary these parameters to achieve a particular stringency ofhybridization.

In general, “stringent hybridization” is performed at about 25° C. belowthe thermal melting point (T_(m)) for the specific DNA hybrid under aparticular set of conditions. “Stringent washing” is performed attemperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., supra, page 9.51, herebyincorporated by reference. For purposes herein, “high stringencyconditions” are defined for solution phase hybridization as aqueoushybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours,followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. Itwill be appreciated by the skilled artisan that hybridization at 65° C.will occur at different rates depending on a number of factors includingthe length and percent identity of the sequences which are hybridizing.

The term “mutated” when applied to nucleic acid sequences means thatnucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product. See, e.g., Leung, D. W., et al., Technique, 1, pp.11-15 (1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2,pp. 28-33 (1992)); and “oligonucleotide-directed mutagenesis” (a processwhich enables the generation of site-specific mutations in any clonedDNA segment of interest. See, e.g., Reidhaar-Olson, J. F. & Sauer, R.T., et al., Science, 241, pp. 53-57 (1988)).

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Other vectors include cosmids, bacterial artificialchromosomes (BAC) and yeast artificial chromosomes (YAC). Another typeof vector is a viral vector, wherein additional DNA segments may beligated into the viral genome (discussed in more detail below). Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., vectors having an origin of replication whichfunctions in the host cell). Other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and arethereby replicated along with the host genome. Moreover, certainpreferred vectors are capable of directing the expression of genes towhich they are operatively linked. Such vectors are referred to hereinas “recombinant expression vectors” (or simply, “expression vectors”).

“Operatively linked” expression control sequences refers to a linkage inwhich the expression control sequence is contiguous with the gene ofinterest to control the gene of interest, as well as expression controlsequences that act in trans or at a distance to control the gene ofinterest.

The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a nucleic acid such asa recombinant vector has been introduced. It should be understood thatsuch terms are intended to refer not only to the particular subject cellbut to the progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” as used herein encompasses bothnaturally-occurring and non-naturally-occurring proteins, and fragments,mutants, derivatives and analogs thereof A polypeptide may be monomericor polymeric. Further, a polypeptide may comprise a number of differentdomains each of which has one or more distinct activities.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) when it exists in a purity not found in nature,where purity can be adjudged with respect to the presence of othercellular material (e.g., is free of other proteins from the samespecies) (3) is expressed by a cell from a different species, or (4)does not occur in nature (e.g., it is a fragment of a polypeptide foundin nature or it includes amino acid analogs or derivatives not found innature or linkages other than standard peptide bonds). Thus, apolypeptide that is chemically synthesized or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A polypeptide orprotein may also be rendered substantially free of naturally associatedcomponents by isolation, using protein purification techniqueswell-known in the art. As thus defined, “isolated” does not necessarilyrequire that the protein, polypeptide, peptide or oligopeptide sodescribed has been physically removed from its native environment.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion compared toa full-length polypeptide. In a preferred embodiment, the polypeptidefragment is a contiguous sequence in which the amino acid sequence ofthe fragment is identical to the corresponding positions in thenaturally-occurring sequence. Fragments typically are at least 5, 6, 7,8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 aminoacids long, more preferably at least 20 amino acids long, morepreferably at least 25, 30, 35, 40 or 45, amino acids, even morepreferably at least 50 or 60 amino acids long, and even more preferablyat least 70 amino acids long.

A “modified derivative” refers to polypeptides or fragments thereof thatare substantially homologous in primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those wellskilled in the art. A variety of methods for labeling polypeptides andof substituents or labels useful for such purposes are well-known in theart, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands whichcan serve as specific binding pair members for a labeled ligand. Thechoice of label depends on the sensitivity required, ease of conjugationwith the primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well-known in the art. See Ausubelet al., 1992, hereby incorporated by reference.

A “polypeptide mutant” or “mutein” refers to a polypeptide whosesequence contains an insertion, duplication, deletion, rearrangement orsubstitution of one or more amino acids compared to the amino acidsequence of a native or wild type protein. A mutein may have one or moreamino acid point substitutions, in which a single amino acid at aposition has been changed to another amino acid, one or more insertionsand/or deletions, in which one or more amino acids are inserted ordeleted, respectively, in the sequence of the naturally-occurringprotein, and/or truncations of the amino acid sequence at either or boththe amino or carboxy termini. A mutein may have the same but preferablyhas a different biological activity compared to the naturally-occurringprotein. For instance, a mutein may have an increased or decreasedneuron or NgR binding activity. In a preferred embodiment of the presentinvention, a MAG derivative that is a mutein (e.g., in MAG Ig-likedomain 5) has decreased neuronal growth inhibitory activity compared toendogenous or soluble wild-type MAG.

A mutein has at least 70% overall sequence homology to its wild-typecounterpart. Even more preferred are muteins having 80%, 85% or 90%overall sequence homology to the wild-type protein. In an even morepreferred embodiment, a mutein exhibits 95% sequence identity, even morepreferably 97%, even more preferably 98% and even more preferably 99%overall sequence identity. Sequence homology may be measured by anycommon sequence analysis algorithm, such as Gap or Bestfit.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, and other unconventional amino acids may also besuitable components for polypeptides of the present invention. Examplesof unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, s-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences). In a preferred embodiment, a homologousprotein is one that exhibits 60% sequence homology to the wild typeprotein, more preferred is 70% sequence homology. Even more preferredare homologous proteins that exhibit 80%, 85% or 90% sequence homologyto the wild type protein. In a yet more preferred embodiment, ahomologous protein exhibits 95%, 97%, 98% or 99% sequence identity. Asused herein, homology between two regions of amino acid sequence(especially with respect to predicted structural similarities) isinterpreted as implying similarity in function.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (see,e.g., Pearson et al., 1994, herein incorporated by reference).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1.

A preferred algorithm when comparing a inhibitory molecule sequence to adatabase containing a large number of sequences from different organismsis the computer program BLAST (Altschul, S. F. et al. (1990) J. Mol.Biol. 215:403-410; Gish and States (1993) Nature Genet. 3:266-272;Madden, T. L. et al. (1996) Meth. Enzymol. 266:131-141; Altschul, S. F.et al. (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. and Madden, T.L. (1997) Genome Res. 7:649-656), especially blastp or tblastn (Altschulet al., 1997). Preferred parameters for BLASTp are: Expectation value:10 (default); Filter: seg (default); Cost to open a gap: 11 (default);Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Wordsize: 11 (default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,1990, herein incorporated by reference). For example, percent sequenceidentity between amino acid sequences can be determined using FASTA withits default parameters (a word size of 2 and the PAM250 scoring matrix),as provided in GCG Version 6.1, herein incorporated by reference.

The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements from two or more differentproteins. A fusion protein comprises at least 10 contiguous amino acidsfrom a polypeptide of interest, more preferably at least 20 or 30 aminoacids, even more preferably at least 40, 50 or 60 amino acids, yet morepreferably at least 75, 100 or 125 amino acids. Fusion proteins can beproduced recombinantly by constructing a nucleic acid sequence whichencodes the polypeptide or a fragment thereof in-frame with a nucleicacid sequence encoding a different protein or peptide and thenexpressing the fusion protein. Alternatively, a fusion protein can beproduced chemically by crosslinking the polypeptide or a fragmentthereof to another protein.

The term “region” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a region is defined by a contiguous portion of the amino acidsequence of that protein.

The term “domain” as used herein refers to a structure of a biomoleculethat contributes to a known or suspected function of the biomolecule.Domains may be co-extensive with regions or portions thereof; domainsmay also include distinct, non-contiguous regions of a biomolecule.Examples of protein domains include, but are not limited to, an Igdomain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

As used herein, the term “molecule” means any compound, including, butnot limited to, a small molecule, peptide, protein, sugar, nucleotide,nucleic acid, lipid, etc., and such a compound can be natural orsynthetic.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Methods for Producing Host Cells Having Man₅GlcNAc₂ ModifiedOligosaccharides for the Generation of Human-Like N-Glycans

The invention provides a method for producing a glycoprotein havinghuman-like glycosylation in a non-human eukaryotic host cell. Asdescribed in more detail below, a eukaryotic host cell that does notnaturally express, or which is engineered not to express, one or moreenzymes involved in production of high mannose structures is selected asa starting host cell. Such a selected host cell is engineered to expressone or more enzymes or other factors required to produce human-likeglycoproteins. A desired host strain can be engineered one enzyme ormore than one enzyme at a time. In addition, a nucleic acid moleculeencoding one or more enzymes or activities may be used to engineer ahost strain of the invention. Preferably, a library of nucleic acidmolecules encoding potentially useful enzymes (e.g., chimeric enzymescomprising a catalytically active enzyme fragment ligated in-frame to aheterologous subcellular targeting sequence) is created (e.g., byligation of sub-libraries comprising enzymatic fragments and subcellulartargeting sequences), and a strain having one or more enzymes withoptimal activities or producing the most “human-like” glycoproteins maybe selected by transforming target host cells with one or more membersof the library.

In particular, the methods described herein enable one to obtain, invivo, Man₅GlcNAc₂ structures in high yield, at least transiently, forthe purpose of further modifying it to yield complex N-glycans. Asuccessful scheme to obtain suitable Man₅GlcNAc₂ structures inappropriate yields in a host cell, such as a lower eukaryotic organism,generally involves two parallel approaches: (1) reducing high mannosestructures made by endogenous mannosyltransferase activities, if any,and (2) removing 1,2-α-mannose by mannosidases to yield high levels ofsuitable Man₅GlcNAc₂ structures which may be further reacted inside thehost cell to form complex, human-like glycoforms.

Accordingly, a first step involves the selection or creation of aeukaryotic host cell, e.g., a lower eukaryote, capable of producing aspecific precursor structure of Man₅GlcNAc₂ that is able to accept invivo GlcNAc by the action of a GlcNAc transferase I (“GnTI”). In oneembodiment, the method involves making or using a non-human eukaryotichost cell depleted in a 1,6 mannosyltransferase activity with respect tothe N-glycan on a glycoprotein. Preferably, the host cell is depleted inan initiating 1,6 mannosyltransferase activity (see below). Such a hostcell will lack one or more enzymes involved in the production of highmannose structures which are undesirable for producing human-likeglycoproteins.

One or more enzyme activities are then introduced into such a host cellto produce N-glycans within the host cell characterized by having atleast 30 mol % of the Man₅GlcNAc₂ (“Man₅”) carbohydrate structures.Man₅GlcNAc₂ structures are necessary for complex N-glycan formation:Man₅GlcNAc₂ must be formed in vivo in a high yield (e.g., in excess of30%), at least transiently, as subsequent mammalian- and human-likeglycosylation reactions require Man₅GlcNAc₂ or a derivative thereof.

This step also requires the formation of a particular isomeric structureof Man₅GlcNAc₂ within the cell at a high yield. While Man₅GlcNAc₂structures are necessary for complex N-glycan formation, their presenceis by no means sufficient. That is because Man₅GlcNAc₂ may occur indifferent isomeric forms, which may or may not serve as a substrate forGlcNAc transferase I. As most glycosylation reactions are not complete,a particular glycosylated protein generally contains a range ofdifferent carbohydrate structures (i.e. glycoforms) on its surface.Thus, the mere presence of trace amounts (i.e., less than 5%) of aparticular structure like Man₅GlcNAc₂ is of little practical relevancefor producing mammalian- or human-like glycoproteins. It is theformation of a GlcNAc transferase I-accepting Man₅GlcNAc₂ intermediate(FIG. 1B) in high yield (i.e., above 30%), which is required. Theformation of this intermediate is necessary to enable subsequent in vivosynthesis of complex N-glycans on glycosylated proteins of interest(target proteins).

Accordingly, some or all of the Man₅GlcNAc₂ produced by the selectedhost cell must be a productive substrate for enzyme activities along amammalian glycosylation pathway, e.g., can serve as a substrate for aGlcNAc transferase I activity in vivo, thereby forming the human-likeN-glycan intermediate GlcNAcMan₅GlcNAc₂ in the host cell. In a preferredembodiment, at least 10%, more preferably at least 30% and mostpreferably 50% or more of the Man₅GlcNAc₂ intermediate produced in thehost cell of the invention is a productive substrate for GnTI in vivo.It is understood that if, for example, GlcNAcMan₅GlcNAc₂ is produced at10% and Man₅GlcNAc₂ is produced at 25% on a target protein, that thetotal amount of transiently produced Man₅GlcNAc₂ is 35% becauseGlcNAcMan₅GlcNAc₂ is a product of Man₅GlcNAc₂.

One of ordinary skill in the art can select host cells from nature,e.g., existing fungi or other lower eukaryotes that produce significantlevels of Man₅GlcNAc₂ in vivo. As yet, however, no lower eukaryote hasbeen shown to provide such structures in vivo in excess of 1.8% of thetotal N-glycans (see e.g. Maras et al., 1997). Alternatively, such hostcells may be genetically engineered to produce the Man₅GlcNAc₂ structurein vivo. Methods such as those described in U.S. Pat. No. 5,595,900 maybe used to identify the absence or presence of particularglycosyltransferases, mannosidases and sugar nucleotide transporters ina target host cell or organism of interest.

Inactivation of Undesirable Host Cell Glycosylation Enzymes

The methods of the invention are directed to making host cells whichproduce glycoproteins having altered, and preferably human-like,N-glycan structures. In a preferred embodiment, the methods are directedto making host cells in which oligosaccharide precursors are enriched inMan₅GlcNAc₂. Preferably, a eukaryotic host cell is used that does notexpress one or more enzymes involved in the production of high mannosestructures. Such a host cell may be found in nature or may beengineered, e.g., starting with or derived from one of many such mutantsalready described in yeasts. Thus, depending on the selected host cell,one or a number of genes that encode enzymes known to be characteristicof non-human glycosylation reactions will have to be deleted. Such genesand their corresponding proteins have been extensively characterized ina number of lower eukaryotes (e.g., S. cerevisiae, T. reesei, A.nidulans etc.), thereby providing a list of known glycosyltransferasesin lower eukaryotes, their activities and their respective geneticsequence. These genes are likely to be selected from the group ofmannosyltransferases e.g. 1,3 mannosyltransferases (e.g. MNN1 in S.cerevisiae) (Graham, 1991), 1,2 mannosyltransferases (e.g. KTR/KREfamily from S. cerevisiae), 1,6 mannosyltransferases (OCH1 from S.cerevisiae), mannosylphosphate transferases and their regulators (MNN4and MNN6 from S. cerevisiae) and additional enzymes that are involved inaberrant, i.e. non human, glycosylation reactions. Many of these geneshave in fact been deleted individually giving rise to viable phenotypeswith altered glycosylation profiles. Examples are shown in Table 1.

Preferred lower eukaryotic host cells of the invention, as describedherein to exemplify the required manipulation steps, arehypermannosylation-minus (och1) mutants of Pichia pastoris or K. lactis.Like other lower eukaryotes, P. pastoris processes Man₉GlcNAc₂structures in the ER with an α-1,2-mannosidase to yield Man₈GlcNAc₂(FIG. 1A). Through the action of several mannosyltransferases, thisstructure is then converted to hypermannosylated structures(Man_(>9)GlcNAc₂), also known as mannans. In addition, it has been foundthat P. pastoris is able to add non-terminal phosphate groups, throughthe action of mannosylphosphate transferases, to the carbohydratestructure. This differs from the reactions performed in mammalian cells,which involve the removal rather than addition of mannose sugars. It isof particular importance to eliminate the ability of the eukaryotic hostcell, e.g., fungus, to hypermannosylate an existing Man₈GlcNAc₂structure. This can be achieved by either selecting for a host cell thatdoes not hypermannosylate or by genetically engineering such a cell.

Genes that are involved in the hypermannosylation process have beenidentified, e.g., in Pichia pastoris, and by creating mutations in thesegenes, one can reduce the production of “undesirable” glycoforms. Suchgenes can be identified by homology to existing mannosyltransferases ortheir regulators (e.g., OCH1, MNN4, MNN6, MNN1) found in other lowereukaryotes such as C. albicans, Pichia angusta or S. cerevisiae or bymutagenizing the host strain and selecting for a glycosylation phenotypewith reduced mannosylation. Based on homologies amongst knownmannosyltransferases and mannosylphosphate transferases, one may eitherdesign PCR primers (SEQ ID NOS 7, 8, 47, & 4 left to right,respectively, in order of appearance)(examples of which are shown inTable 2), or use genes or gene fragments encoding such enzymes as probesto identify homologs in DNA libraries of the target or a relatedorganism. Alternatively, one may identify a functional homolog havingmannosyltransferase activity by its ability to complement particularglycosylation phenotypes in related organisms.

TABLE 2 PCR Primers Target Gene(s) in PCR primer A PCR primer BP. pastoris Homologs ATGGCGAAGGCAGA TTAGTCCTTCCAAC 1,6-OCH1 S. cerevisiae, TGGCAGT TTCCTTC mannosyltransferase Pichia albicans(SEQ ID (SEQ ID NO: 1) NO: 2) TAYTGGMGNGTNGA GCRTCNCCCCANCK 1,2KTR/KRE family, RCYNGAYATHAA YTCRTA mannosyltransferases S. cerevisiae(SEQ ID (SEQ ID NO: 3) NO: 4) Legend: M = A or C, R = A or G, W = A orT, S = C or G, Y = C or T, K = G or T, V = A or C or G, H = A or C or T,D = A or G or T, B = C or G or T, N = G or A or T or C.

To obtain the gene or genes encoding 1,6-mannosyltransferase activity inP. pastoris, for example, one would carry out the following steps: OCH1mutants of S. cerevisiae are temperature sensitive and are slow growersat elevated temperatures. One can thus identify functional homologs ofOCH1 in P. pastoris by complementing an OCH1 mutant of S. cerevisiaewith a P. pastoris DNA or cDNA library. Mutants of S. cerevisiae areavailable, e.g., from Stanford University and are commercially availablefrom ResGen, an Invitrogen Corp. (Carlsbad, Calif.). Mutants thatdisplay a normal growth phenotype at elevated temperature, after havingbeen transformed with a P. pastoris DNA library, are likely to carry anOCH1 homolog of P. pastoris. Such a library can be created by partiallydigesting chromosomal DNA of P. pastoris with a suitable restrictionenzyme and, after inactivating the restriction enzyme, ligating thedigested DNA into a suitable vector, which has been digested with acompatible restriction enzyme.

Suitable vectors include, e.g., pRS314, a low copy (CEN6/ARS4) plasmidbased on pBluescript containing the Trp1 marker (Sikorski, R. S., andHieter, P., 1989, Genetics 122, pg 19-27) and pFL44S, a high copy (2μ)plasmid based on a modified pUC19 containing the URA3 marker (Bonneaud,N., et al., 1991, Yeast 7, pg. 609-615). Such vectors are commonly usedby academic researchers and similar vectors are available from a numberof different vendors (e.g., Invitrogen (Carlsbad, Calif.); Pharmacia(Piscataway, N.J.); New England Biolabs (Beverly, Mass.)). Furtherexamples include pYES/GS, 2μ origin of replication based yeastexpression plasmid from Invitrogen, or Yep24 cloning vehicle from NewEngland Biolabs.

After ligation of the chromosomal DNA and the vector, one may transformthe DNA library into a strain of S. cerevisiae with a specific mutationand select for the correction of the corresponding phenotype. Aftersub-cloning and sequencing the DNA fragment that is able to restore thewild-type phenotype, one may use this fragment to eliminate the activityof the gene product encoded by OCH1 in P. pastoris using in vivomutagenesis and/or recombination techniques well-known to those skilledin the art.

Alternatively, if the entire genomic sequence of a particular host cell,e.g., fungus, of interest is known, one may identify such genes simplyby searching publicly available DNA databases, which are available fromseveral sources, such as NCBI, Swissprot. For example, by searching agiven genomic sequence or database with sequences from a known 1,6mannosyltransferase gene (e.g., OCH1 from S. cerevisiae), one canidentify genes of high homology in such a host cell genome which may(but do not necessarily) encode proteins that have1,6-mannosyltransferase activity. Nucleic acid sequence homology aloneis not enough to prove, however, that one has identified and isolated ahomolog encoding an enzyme having the same activity. To date, forexample, no data exist to show that an OCH1 deletion in P. pastoriseliminates the crucial initiating 1,6-mannosyltransferase activity.(Martinet et al. Biotech. Letters 20(12) (December 1998): 1171-1177;Contreras et al. WO 02/00856 A2). Thus, no data prove that the P.pastoris OCH1 gene homolog actually encodes that function. Thatdemonstration is provided for the first time herein.

Homologs to several S. cerevisiae mannosyltransferases have beenidentified in P. pastoris using these approaches. Homologous genes oftenhave similar functions to genes involved in the mannosylation ofproteins in S. cerevisiae and thus their deletion may be used tomanipulate the glycosylation pattern in P. pastoris or, by analogy, inany other host cell, e.g., fungus, plant, insect or animal cells, withsimilar glycosylation pathways.

The creation of gene knock-outs, once a given target gene sequence hasbeen determined, is a well-established technique in the art and can becarried out by one of ordinary skill in the art (see, e.g., R.Rothstein, (1991) Methods in Enzymology, vol. 194, p. 281). The choiceof a host organism may be influenced by the availability of goodtransformation and gene disruption techniques.

If several mannosyltransferases are to be knocked out, the methoddeveloped by Alani and Kleckner, (Genetics 116:541-545 (1987)), forexample, enables the repeated use of a selectable marker, e.g., the URA3marker in yeast, to sequentially eliminate all undesirable endogenousmannosyltransferase activity. This technique has been refined by othersbut basically involves the use of two repeated DNA sequences, flanking acounter selectable marker. For example: URA3 may be used as a marker toensure the selection of a transformants that have integrated aconstruct. By flanking the URA3 marker with direct repeats one may firstselect for transformants that have integrated the construct and havethus disrupted the target gene. After isolation of the transformants,and their characterization, one may counter select in a second round forthose that are resistant to 5-fluoroorotic acid (5-FOA). Colonies thatare able to survive on plates containing 5-FOA have lost the URA3 markeragain through a crossover event involving the repeats mentioned earlier.This approach thus allows for the repeated use of the same marker andfacilitates the disruption of multiple genes without requiringadditional markers. Similar techniques for sequential elimination ofgenes adapted for use in another eukaryotic host cells with otherselectable and counter-selectable markers may also be used.

Eliminating specific mannosyltransferases, such as 1,6mannosyltransferase (OCH1) or mannosylphosphate transferases (MNN6, orgenes complementing lbd mutants) or regulators (MNN4) in P. pastorisenables one to create engineered strains of this organism whichsynthesize primarily Man₈GlcNAc₂ and which can be used to further modifythe glycosylation pattern to resemble more complex glycoform structures,e.g., those produced in mammalian, e.g., human cells. A preferredembodiment of this method utilizes DNA sequences encoding biochemicalglycosylation activities to eliminate similar or identical biochemicalfunctions in P. pastoris to modify the glycosylation structure ofglycoproteins produced in the genetically altered P. pastoris strain.

Methods used to engineer the glycosylation pathway in yeasts asexemplified herein can be used in filamentous fungi to produce apreferred substrate for subsequent modification. Strategies formodifying glycosylation pathways in A. niger and other filamentousfungi, for example, can be developed using protocols analogous to thosedescribed herein for engineering strains to produce human-likeglycoproteins in yeast. Undesired gene activities involved in 1,2mannosyltransferase activity, e.g., KTR/KRE homologs, are modified oreliminated. A filamentous fungus, such as Aspergillus, is a preferredhost because it lacks the 1,6 mannosyltransferase activity and as such,one would not expect a hypermannosylating gene activity, e.g. OCH1, inthis host. By contrast, other desired activities (e.g.,α-1,2-mannosidase, UDP-GlcNAc transporter, glycosyltransferase (GnT),galactosyltransferase (GalT) and sialyltransferase (ST)) involved inglycosylation are introduced into the host using the targeting methodsof the invention.

Engineering or Selecting Hosts Having Diminished Initiating α-1,6Mannosyltransferase Activity

In a preferred embodiment, the method of the invention involves makingor using a host cell which is diminished or depleted in the activity ofan initiating α-1,6-mannosyltransferase, i.e., an initiation specificenzyme that initiates outer chain mannosylation on the α-1,3 arm of theMan₃GlcNAc₂ core structure. In S. cerevisiae, this enzyme is encoded bythe OCH1 gene. Disruption of the OCH1 gene in S. cerevisiae results in aphenotype in which N-linked sugars completely lack the poly-mannoseouter chain. Previous approaches for obtaining mammalian-typeglycosylation in fungal strains have required inactivation of OCH1 (see,e.g., Chiba, 1998). Disruption of the initiatingα-1,6-mannosyltransferase activity in a host cell of the invention maybe optional, however (depending on the selected host cell), as the Och1penzyme requires an intact Man₈GlcNAc₂ for efficient mannose outer chaininitiation. Thus, host cells selected or produced according to thisinvention which accumulate oligosaccharides having seven or fewermannose residues may produce hypoglycosylated N-glycans that will likelybe poor substrates for Och1p (see, e.g., Nakayama, 1997).

The OCH1 gene was cloned from P. pastoris (Example 1) and K. lactis(Example 16), as described. The nucleic acid and amino acid sequences ofthe OCH1 gene from K. lactis are set forth in SEQ ID NOS: 41 and 42.Using gene-specific primers, a construct was made from each clone todelete the OCH1 gene from the genome of P. pastoris and K. lactis(Examples 1 and 16, respectively). Host cells depleted in initiatingα-1,6-mannosyltransferase activity and engineered to produce N-glycanshaving a Man₅GlcNAc₂ carbohydrate structure were thereby obtained (see,e.g., FIGS. 5 and 6; Examples 11 and 16).

Thus, in another embodiment, the invention provides an isolated nucleicacid molecule having a nucleic acid sequence comprising or consisting ofat least forty-five, preferably at least 50, more preferably at least 60and most preferably 75 or more nucleotide residues of the K. lactis OCH1gene (SEQ ID NO: 41), and homologs, variants and derivatives thereof.The invention also provides nucleic acid molecules that hybridize understringent conditions to the above-described nucleic acid molecules.Similarly, isolated polypeptides (including muteins, allelic variants,fragments, derivatives, and analogs) encoded by the nucleic acidmolecules of the invention are provided. Also provided are vectors,including expression vectors, which comprise the above nucleic acidmolecules of the invention, as described further herein. Similarly, hostcells transformed with the nucleic acid molecules or vectors of theinvention are provided.

Host Cells of the Invention

A preferred host cell of the invention is a lower eukaryotic cell, e.g.,yeast, a unicellular and multicellular or filamentous fungus. However, awide variety of host cells are envisioned as being useful in the methodsof the invention. Plant cells or insect cells, for instance, may beengineered to express a human-like glycoprotein according to theinvention (Examples 17 and 18). Likewise, a variety of non-human,mammalian host cells may be altered to express more human-like orotherwise altered glycoproteins using the methods of the invention. Asone of skill in the art will appreciate, any eukaryotic host cell(including a human cell) may be used in conjunction with a library ofthe invention to express one or more chimeric proteins which is targetedto a subcellular location, e.g., organelle, in the host cell where theactivity of the protein is modified, and preferably is enhanced. Such aprotein is preferably—but need not necessarily be—an enzyme involved inprotein glycosylation, as exemplified herein. It is envisioned that anyprotein coding sequence may be targeted and selected for modifiedactivity in a eukaryotic host cell using the methods described herein.

Lower eukaryotes that are able to produce glycoproteins having theattached N-glycan Man₅GlcNAc₂ are particularly useful because (a)lacking a high degree of mannosylation (e.g. greater than 8 mannoses perN-glycan, or especially 30-40 mannoses), they show reducedimmunogenicity in humans; and (b) the N-glycan is a substrate forfurther glycosylation reactions to form an even more human-likeglycoform, e.g., by the action of GlcNAc transferase I (FIG. 1B; β1,2GnTI) to form GlcNAcMan₅GlcNAc₂. A yield is obtained of greater than 30mole %, more preferably a yield of 50-100 mole %, glycoproteins withN-glycans having a Man₅GlcNAc₂ structure. In a preferred embodiment,more than 50% of the Man₅GlcNAc₂ structure is shown to be a substratefor a GnTI activity and can serve as such a substrate in vivo.

Preferred lower eukaryotes of the invention include but are not limitedto: Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichiakoclamae, Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reseei, Chrysosporiumlucknowense, Fusarium sp. Fusarium gramineum, Fusarium venenatum andNeurospora crassa.

In each above embodiment, the method is directed to making a host cellin which the oligosaccharide precursors are enriched in Man₅GlcNAc₂.These structures are desirable because they may then be processed bytreatment in vitro, for example, using the method of Maras andContreras, U.S. Pat. No. 5,834,251. In a preferred embodiment, however,precursors enriched in Man₅GlcNAc₂ are processed by at least one furtherglycosylation reaction in vivo—with glycosidases (e.g., α-mannosidases)and glycosyltransferases (e.g., GnTI)—to produce human-like N-glycans.Oligosaccharide precursors enriched in Man₅GlcNAc₂, for example, arepreferably processed to those having GlcNAcMan_(x)GlcNAc₂ corestructures, wherein X is 3, 4 or 5, and is preferably 3. N-glycanshaving a GlcNAcMan_(x)GlcNAc₂ core structure where X is greater than 3may be converted to GlcNAcMan₃GlcNAc₂, e.g., by treatment with an α-1,3and/or α-1,6 mannosidase activity, where applicable. Additionalprocessing of GlcNAcMan₃GlcNAc₂ by treatment with glycosyltransferases(e.g., GnTII) produces GlcNAc₂Man₃GlcNAc₂ core structures which may thenbe modified, as desired, e.g., by ex vivo treatment or by heterologousexpression in the host cell of additional glycosylation enzymes,including glycosyltransferases, sugar transporters and mannosidases (seebelow), to become human-like N-glycans.

Preferred human-like glycoproteins which may be produced according tothe invention include those which comprise N-glycans having seven orfewer, or three or fewer, mannose residues; and which comprise one ormore sugars selected from the group consisting of galactose, GlcNAc,sialic acid, and fucose.

Formation of Complex N-Glycans

Formation of complex N-glycan synthesis is a sequential process by whichspecific sugar residues are removed and attached to the coreoligosaccharide structure. In higher eukaryotes, this is achieved byhaving the substrate sequentially exposed to various processing enzymes.These enzymes carry out specific reactions depending on their particularlocation within the entire processing cascade. This “assembly line”consists of ER, early, medial and late Golgi, and the trans Golginetwork all with their specific processing environment. To re-create theprocessing of human glycoproteins in the Golgi and ER of lowereukaryotes, numerous enzymes (e.g. glycosyltransferases, glycosidases,phosphatases and transporters) have to be expressed and specificallytargeted to these organelles, and preferably, in a location so that theyfunction most efficiently in relation to their environment as well as toother enzymes in the pathway.

Because one goal of the methods described herein is to achieve a robustprotein production strain that is able to perform well in an industrialfermentation process, the integration of multiple genes into the hostcell chromosome involves careful planning. As described above, one ormore genes which encode enzymes known to be characteristic of non-humanglycosylation reactions are preferably deleted. The engineered cellstrain is transformed with a range of different genes encoding desiredactivities, and these genes are transformed in a stable fashion, therebyensuring that the desired activity is maintained throughout thefermentation process.

Any combination of the following enzyme activities may be engineeredsingly or multiply into the host using methods of the invention:sialyltransferases, mannosidases, fucosyltransferases,galactosyltransferases, GlcNAc transferases, ER and Golgi specifictransporters (e.g. syn- and antiport transporters for UDP-galactose andother precursors), other enzymes involved in the processing ofoligosaccharides, and enzymes involved in the synthesis of activatedoligosaccharide precursors such as UDP-galactose andCMP-N-acetylneuraminic acid. Preferably, enzyme activities areintroduced on one or more nucleic acid molecules (see also below).Nucleic acid molecules may be introduced singly or multiply, e.g., inthe context of a nucleic acid library such as a combinatorial library ofthe invention. It is to be understood, however, that single or multipleenzymatic activities may be introduced into a host cell in any fashion,including but not limited to protein delivery methods and/or by use ofone or more nucleic acid molecules without necessarily using a nucleicacid library or combinatorial library of the invention.

Expression of Glycosyltransferases to Produce Complex N-Glycans:

With DNA sequence information, the skilled artisan can clone DNAmolecules encoding GnT activities (e.g., Examples 3 and 4). Usingstandard techniques well-known to those of skill in the art, nucleicacid molecules encoding GnTI, II, III, IV or V (or encodingcatalytically active fragments thereof) may be inserted into appropriateexpression vectors under the transcriptional control of promoters andother expression control sequences capable of driving transcription in aselected host cell of the invention, e.g., a fungal host such as Pichiasp., Kluyveromyces sp. and Aspergillus sp., as described herein, suchthat one or more of these mammalian GnT enzymes may be activelyexpressed in a host cell of choice for production of a human-likecomplex glycoprotein (e.g., Examples 15, 17 and 18).

Several individual glycosyltransferases have been cloned and expressedin S. cerevisiae (GalT, GnTI), Aspergillus nidulans (GnTI) and otherfungi, without however demonstrating the desired outcome of“humanization” on the glycosylation pattern of the organisms (Yoshida,1995; Schwientek, 1995; Kalsner, 1995). It was speculated that thecarbohydrate structure required to accept sugars by the action of suchglycosyltransferases was not present in sufficient amounts, which mostlikely contributed to the lack of complex N-glycan formation.

A preferred method of the invention provides the functional expressionof a GnT, such as GnTI, in the early or medial Golgi apparatus as wellas ensuring a sufficient supply of UDP-GlcNAc (e.g., by expression of aUDP-GlcNAc transporter; see below).

Methods for Providing Sugar Nucleotide Precursors to the GolgiApparatus:

For a glycosyltransferase to function satisfactorily in the Golgi, theenzyme requires a sufficient concentration of an appropriate nucleotidesugar, which is the high-energy donor of the sugar moiety added to anascent glycoprotein. In humans, the full range of nucleotide sugarprecursors (e.g. UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine,CMP-N-acetylneuraminic acid, UDP-galactose, etc.) are generallysynthesized in the cytosol and transported into the Golgi, where theyare attached to the core oligosaccharide by glycosyltransferases.

To replicate this process in non-human host cells such as lowereukaryotes, sugar nucleoside specific transporters have to be expressedin the Golgi to ensure adequate levels of nucleoside sugar precursors(Sommers, 1981; Sommers, 1982; Perez, 1987). Nucleotide sugars may beprovided to the appropriate compartments, e.g., by expressing in thehost microorganism an exogenous gene encoding a sugar nucleotidetransporter. The choice of transporter enzyme is influenced by thenature of the exogenous glycosyltransferase being used. For example, aGlcNAc transferase may require a UDP-GlcNAc transporter, afucosyltransferase may require a GDP-fucose transporter, agalactosyltransferase may require a UDP-galactose transporter, and asialyltransferase may require a CMP-sialic acid transporter.

The added transporter protein conveys a nucleotide sugar from thecytosol into the Golgi apparatus, where the nucleotide sugar may bereacted by the glycosyltransferase, e.g. to elongate an N-glycan. Thereaction liberates a nucleoside diphosphate or monophosphate, e.g. UDP,GDP, or CMP. Nucleoside monophosphates can be directly exported from theGolgi in exchange for nucleoside triphosphate sugars by an antiportmechanism. Accumulation of a nucleoside diphosphate, however, inhibitsthe further activity of a glycosyltransferase. As this reaction appearsto be important for efficient glycosylation, it is frequently desirableto provide an expressed copy of a gene encoding a nucleotidediphosphatase. The diphosphatase (specific for UDP or GDP asappropriate) hydrolyzes the diphosphonucleoside to yield a nucleosidemonosphosphate and inorganic phosphate.

Suitable transporter enzymes, which are typically of mammalian origin,are described below. Such enzymes may be engineered into a selected hostcell using the methods of the invention (see also Examples 7-10).

In another example, α2,3- or α2,6-sialyltransferase caps galactoseresidues with sialic acid in the trans-Golgi and TGN of humans leadingto a mature form of the glycoprotein (FIG. 1B). To reengineer thisprocessing step into a metabolically engineered yeast or fungus willrequire (1) α2,3- or α2,6-sialyltransferase activity and (2) asufficient supply of CMP-N-acetyl neuraminic acid, in the late Golgi ofyeast (Example 6). To obtain sufficient α2,3-sialyltransferase activityin the late Golgi, for example, the catalytic domain of a knownsialyltransferase (e.g. from humans) has to be directed to the lateGolgi in fungi (see above). Likewise, transporters have to be engineeredto allow the transport of CMP-N-acetyl neuraminic acid into the lateGolgi. There is currently no indication that fungi synthesize or caneven transport sufficient amounts of CMP-N-acetyl neuraminic acid intothe Golgi. Consequently, to ensure the adequate supply of substrate forthe corresponding glycosyltransferases, one has to metabolicallyengineer the production of CMP-sialic acid into the fungus.

UDP-N-Acetylglucosamine

The cDNA of human UDP-N-acetylglucosamine transporter, which wasrecognized through a homology search in the expressed sequence tagsdatabase (dbEST), has been cloned (Ishida, 1999 J. Biochem. 126(1):68-77). The mammalian Golgi membrane transporter forUDP-N-acetylglucosamine was cloned by phenotypic correction with cDNAfrom canine kidney cells (MDCK) of a recently characterizedKluyveromyces lactic mutant deficient in Golgi transport of the abovenucleotide sugar (Guillen, 1998). Results demonstrate that the mammalianGolgi UDP-GlcNAc transporter gene has all of the necessary informationfor the protein to be expressed and targeted functionally to the Golgiapparatus of yeast and that two proteins with very different amino acidsequences may transport the same solute within the same Golgi membrane(Guillen, 1998).

Accordingly, one may incorporate the expression of a UDP-GlcNActransporter in a host cell by means of a nucleic acid construct whichmay contain, for example: (1) a region by which the transformedconstruct is maintained in the cell (e.g. origin of replication or aregion that mediates chromosomal integration), (2) a marker gene thatallows for the selection of cells that have been transformed, includingcounterselectable and recyclable markers such as ura3 or T-urf13(Soderholm, 2001) or other well characterized selection-markers (e.g.,his4, bla, Sh ble etc.), (3) a gene or fragment thereof encoding afunctional UDP-GlcNAc transporter (e.g. from K. lactis, (Abeijon, (1996)Proc. Natl. Acad. Sci. 93:5963-5968), or from H. sapiens (Ishida, 1996),and (4) a promoter activating the expression of the above mentionedlocalization/catalytic domain fusion construct library.

GDP-Fucose

The rat liver Golgi membrane GDP-fucose transporter has been identifiedand purified by Puglielli, L. and C. B. Hirschberg (Puglielli, 1999 J.Biol. Chem. 274(50):35596-35600). The corresponding gene has not beenidentified, however, N-terminal sequencing can be used for the design ofoligonucleotide probes specific for the corresponding gene. Theseoligonucleotides can be used as probes to clone the gene encoding forGDP-fucose transporter.

UDP-Galactose

Two heterologous genes, gmal2(+) encoding alpha1,2-galactosyltransferase (alpha 1,2 GalT) from Schizosaccharomycespombe and (hUGT2) encoding human UDP-galactose (UDP-Gal) transporter,have been functionally expressed in S. cerevisiae to examine theintracellular conditions required for galactosylation. Correlationbetween protein galactosylation and UDP-galactose transport activityindicated that an exogenous supply of UDP-Gal transporter, rather thanalpha 1,2 GalT played a key role for efficient galactosylation in S.cerevisiae (Kainuma, 1999 Glycobiology 9(2): 133-141). Likewise, anUDP-galactose transporter from S. pombe was cloned (Aoki, 1999 J.Biochem. 126(5): 940-950; Segawa, 1999 Febs Letters 451(3): 295-298).

CMP-N-Acetylneuraminic Acid (CMP-Sialic Acid).

Human CMP-sialic acid transporter (hCST) has been cloned and expressedin Lec 8 CHO cells (Aoki, 1999; Eckhardt, 1997). The functionalexpression of the murine CMP-sialic acid transporter was achieved inSaccharomyces cerevisiae (Berninsone, 1997). Sialic acid has been foundin some fungi, however it is not clear whether the chosen host systemwill be able to supply sufficient levels of CMP-Sialic acid. Sialic acidcan be either supplied in the medium or alternatively fungal pathwaysinvolved in sialic acid synthesis can also be integrated into the hostgenome.

Expression of Diphosphatases:

When sugars are transferred onto a glycoprotein, either a nucleosidediphosphate or monophosphate is released from the sugar nucleotideprecursors. While monophosphates can be directly exported in exchangefor nucleoside triphosphate sugars by an antiport mechanism,diphosphonucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g.GDPase) to yield nucleoside monophosphates and inorganic phosphate priorto being exported. This reaction appears to be important for efficientglycosylation, as GDPase from S. cerevisiae has been found to benecessary for mannosylation. However, the enzyme only has 10% of theactivity towards UDP (Berninsone, 1994). Lower eukaryotes often do nothave UDP-specific diphosphatase activity in the Golgi as they do notutilize UDP-sugar precursors for glycoprotein synthesis in the Golgi.Schizosaccharomyces pombe, a yeast which adds galactose residues to cellwall polysaccharides (from UDP-galactose), was found to have specificUDPase activity, further suggesting the requirement for such an enzyme(Berninsone, 1994). UDP is known to be a potent inhibitor ofglycosyltransferases and the removal of this glycosylation side productis important to prevent glycosyltransferase inhibition in the lumen ofthe Golgi (Khatara et al. 1974).

Methods for Altering N-Glycans in a Host by Expressing a TargetedEnzymatic Activity From a Nucleic Acid Molecule

The present invention further provides a method for producing ahuman-like glycoprotein in a non-human host cell comprising the step ofintroducing into the cell one or more nucleic acid molecules whichencode an enzyme or enzymes for production of the Man₅GlcNAc₂carbohydrate structure. In one preferred embodiment, a nucleic acidmolecule encoding one or more mannosidase activities involved in theproduction of Man₅GlcNAc₂ from Man₈GlcNAc₂ or Man₉GlcNAc₂ is introducedinto the host. The invention additionally relates to methods for makingaltered glycoproteins in a host cell comprising the step of introducinginto the host cell a nucleic acid molecule which encodes one or moreglycosylation enzymes or activities. Preferred enzyme activities areselected from the group consisting of UDP-GlcNAc transferase,UDP-galactosyltransferase, GDP-fucosyltransferase,CMP-sialyltransferase, UDP-GlcNAc transporter, UDP-galactosetransporter, GDP-fucose transporter, CMP-sialic acid transporter, andnucleotide diphosphatases. In a particularly preferred embodiment, thehost is selected or engineered to express two or more enzymaticactivities in which the product of one activity increases substratelevels of another activity, e.g., a glycosyltransferase and acorresponding sugar transporter, e.g., GnTI and UDP-GlcNAc transporteractivities. In another preferred embodiment, the host is selected orengineered to expresses an activity to remove products which may inhibitsubsequent glycosylation reactions, e.g. a UDP- or GDP-specificdiphosphatase activity.

Preferred methods of the invention involve expressing one or moreenzymatic activities from a nucleic acid molecule in a host cell andcomprise the step of targeting at least one enzymatic activity to adesired subcellular location (e.g., an organelle) by forming a fusionprotein comprising a catalytic domain of the enzyme and a cellulartargeting signal peptide, e.g., a heterologous signal peptide which isnot normally ligated to or associated with the catalytic domain. Thefusion protein is encoded by at least one genetic construct (“fusionconstruct”) comprising a nucleic acid fragment encoding a cellulartargeting signal peptide ligated in the same translational reading frame(“in-frame”) to a nucleic acid fragment encoding an enzyme (e.g.,glycosylation enzyme), or catalytically active fragment thereof.

The targeting signal peptide component of the fusion construct orprotein is preferably derived from a member of the group consisting of:membrane-bound proteins of the ER or Golgi, retrieval signals, Type IImembrane proteins, Type I membrane proteins, membrane spanningnucleotide sugar transporters, mannosidases, sialyltransferases,glucosidases, mannosyltransferases and phosphomannosyltransferases.

The catalytic domain component of the fusion construct or protein ispreferably derived from a glycosidase, mannosidase or aglycosyltransferase activity derived from a member of the groupconsisting of GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI, GalT,Fucosyltransferase and Sialyltransferase. The catalytic domainpreferably has a pH optimum within 1.4 pH units of the average pHoptimum of other representative enzymes in the organelle in which theenzyme is localized, or has optimal activity at a pH between 5.1 and8.0. In a preferred embodiment, the catalytic domain encodes amannosidase selected from the group consisting of C. elegans mannosidaseIA, C. elegans mannosidase IB, D. melanogaster mannosidase IA, H.sapiens mannosidase IB, P. citrinum mannosidase I, mouse mannosidase IA,mouse mannosidase IB, A. nidulans mannosidase IA, A. nidulansmannosidase IB, A. nidulans mannosidase IC, mouse mannosidase II, C.elegans mannosidase II, H. sapiens mannosidase II, and mannosidase III.

Selecting a Glycosylation Enzyme: pH Optima and Subcellular Localization

In one embodiment of the invention, a human-like glycoprotein is madeefficiently in a non-human eukaryotic host cell by introducing into asubcellular compartment of the cell a glycosylation enzyme selected tohave a pH optimum similar to the pH optima of other enzymes in thetargeted subcellular compartment. For example, most enzymes that areactive in the ER and Golgi apparatus of S. cerevisiae have pH optimathat are between about 6.5 and 7.5 (see Table 3). Because theglycosylation of proteins is a highly evolved and efficient process, theinternal pH of the ER and the Golgi is likely also in the range of about6-8. All previous approaches to reduce mannosylation by the action ofrecombinant mannosidases in fungal hosts, however, have introducedenzymes that have a pH optimum of around pH 5.0 (Martinet et al., 1998,and Chiba et al., 1998). At pH 7.0, the in vitro determined activity ofthose mannosidases is reduced to less than 10%, which is likelyinsufficient activity at their point of use, namely, the ER and earlyGolgi, for the efficient in vivo production of Man₅GlcNAc₂ on N-glycans.

Accordingly, a preferred embodiment of this invention targets a selectedglycosylation enzyme (or catalytic domain thereof), e.g., anα-mannosidase, to a subcellular location in the host cell (e.g., anorganelle) where the pH optimum of the enzyme or domain is within 1.4 pHunits of the average pH optimum of other representative marker enzymeslocalized in the same organelle(s). The pH optimum of the enzyme to betargeted to a specific organelle should be matched with the pH optimumof other enzymes found in the same organelle to maximize the activityper unit enzyme obtained. Table 3 summarizes the activity ofmannosidases from various sources and their respective pH optima. Table4 summarizes their typical subcellular locations.

TABLE 3 Mannosidases and their pH optimum. pH Source Enzyme optimumReference Aspergillus α-1,2-mannosidase 5.0 Ichishima et al. , saitoi1999 Biochem. J. 339(Pt 3): 589-597 Trichoderma α-1,2-mannosidase 5.0Maras et al. , 2000 reesei J. Biotechnol. 77 (2-3): 255-263 Penicilliumα-D-1,2- 5.0 Yoshida et al. , citrinum mannosidase 1993 Biochem. J.290(Pt 2): 349-354 C. elegans α-1,2-mannosidase 5.5 see FIG. 11Aspergillus α-1,2-mannosidase 6.0 Eades and Hintz, nidulans 2000 Homosapiens α-1,2-mannosidase 6.0 IA(Golgi) Homo sapiens α-1,2-mannosidase6.0 IB (Golgi) Lepidopteran Type I α-1,2-Man₆- 6.0 Ren et al. , 1995insect cells mannosidase Biochem. 34(8): 2489-2495 Homo sapiensα-D-mannosidase 6.0 Chandrasekaran et al. ,1984 Cancer Res. 44(9):4059-68 Xanthomonas α-1,2,3- 6.0 U.S. Pat. No. manihotis mannosidase 6,300, 113 Mouse IB α-1,2-mannosidase 6.5 Schneikert and (Golgi)Herscovics, 1994 Glycobiology. 4 (4): 445-50 Bacillus sp. α-D-1,2- 7.0Maruyama et al. , (secreted) mannosidase 1994 Carbohydrate Res. 251:89-98

In a preferred embodiment, a particular enzyme or catalytic domain istargeted to a subcellular location in the host cell by means of achimeric fusion construct encoding a protein comprising a cellulartargeting signal peptide not normally associated with the enzymaticdomain. Preferably, an enzyme or domain is targeted to the ER, theearly, medial or late Golgi of the trans Golgi apparatus of the hostcell.

In a more preferred embodiment, the targeted glycosylation enzyme is amannosidase, glycosyltransferase or a glycosidase. In an especiallypreferred embodiment, mannosidase activity is targeted to the ER or cisGolgi, where the early reactions of glycosylation occur. While thismethod is useful for producing a human-like glycoprotein in a non-humanhost cell, it will be appreciated that the method is also useful moregenerally for modifying carbohydrate profiles of a glycoprotein in anyeukaryotic host cell, including human host cells.

Targeting sequences which mediate retention of proteins in certainorganelles of the host cell secretory pathway are well-known anddescribed in the scientific literature and public databases, asdiscussed in more detail below with respect to libraries for selectionof targeting sequences and targeted enzymes. Such subcellular targetingsequences may be used alone or in combination to target a selectedglycosylation enzyme (or catalytic domain thereof) to a particularsubcellular location in a host cell, i.e., especially to one where theenzyme will have enhanced or optimal activity based on pH optima or thepresence of other stimulatory factors.

When one attempts to trim high mannose structures to yield Man₅GlcNAc₂in the ER or the Golgi apparatus of a host cell such as S. cerevisiae,for example, one may choose any enzyme or combination of enzymes that(1) has a sufficiently close pH optimum (i.e. between pH 5.2 and pH7.8), and (2) is known to generate, alone or in concert, the specificisomeric Man₅GlcNAc₂ structure required to accept subsequent addition ofGlcNAc by GnTI. Any enzyme or combination of enzymes that is shown togenerate a structure that can be converted to GlcNAcMan₅GlcNAc₂ by GnTIin vitro would constitute an appropriate choice. This knowledge may beobtained from the scientific literature or experimentally.

For example, one may determine whether a potential mannosidase canconvert Man₈GlcNAc₂-2AB (2-aminobenzamide) to Man₅GlcNAc₂-AB and thenverify that the obtained Man₅GlcNAc₂-2AB structure can serve a substratefor GnTI and UDP-GlcNAc to give GlcNAcMan₅GlcNAc₂ in vitro. MannosidaseIA from a human or murine source, for example, would be an appropriatechoice (see, e.g., Example 11). Examples described herein utilize2-aminobenzamide labeled N-linked oligomannose followed by HPLC analysisto make this determination.

TABLE 4 Cellular location and pH optima of various glycosylation-relatedenzymes of S.cerevisiae. pH Gene Activity Location optimum Reference(s)KTR1 α-1,2 Golgi 7.0 Romero et al. , 1997 mannosyltransferase Biochem.J. 321 (Pt 2): 289-295 MNS1 α-1,2-mannosidase ER 6.5 CWH41 glucosidase IER 6.8 — mannosyltransferase Golgi 7-8 Lehele and Tanner, 1974 Biochim.Biophys. Acta 350(1): 225-235 KRE2 α-1,2 Golgi 6.5-9.0 Romero et al. ,mannosyltransferase 1997

Accordingly, a glycosylation enzyme such as an α-1,2-mannosidase enzymeused according to the invention has an optimal activity at a pH ofbetween 5.1 and 8.0. In a preferred embodiment, the enzyme has anoptimal activity at a pH of between 5.5 and 7.5. The C. elegansmannosidase enzyme, for example, works well in the methods of theinvention and has an apparent pH optimum of about 5.5). Preferredmannosidases include those listed in Table 3 having appropriate pHoptima, e.g. Aspergillus nidulans, Homo sapiens IA (Golgi), Homo sapiensIB (Golgi), Lepidopteran insect cells (IPLB-SF21AE), Homo sapiens, mouseIB (Golgi), Xanthomonas manihotis, Drosophila melanogaster and C.elegans.

The experiment which illustrates the pH optimum for an α-1,2-mannosidaseenzyme is described in Example 14. A chimeric fusion protein BB27-2(Saccharomyces MNN10 (s)/C. elegans mannosidase IB Δ31), which leaksinto the medium was subjected to various pH ranges to determine theoptimal activity of the enzyme. The results of the experiment show thatthe α-1,2-mannosidase has an optimal pH of about 5.5 for its function(FIG. 11).

In a preferred embodiment, a single cloned mannosidase gene is expressedin the host organism. However, in some cases it may be desirable toexpress several different mannosidase genes, or several copies of oneparticular gene, in order to achieve adequate production of Man₅GlcNAc₂.In cases where multiple genes are used, the encoded mannosidasespreferably all have pH optima within the preferred range of about 5.1 toabout 8.0, or especially between about 5.5 and about 7.5. Preferredmannosidase activities include α-1,2-mannosidases derived from mouse,human, Lepidoptera, Aspergillus nidulans, or Bacillus sp., C. elegans,D. melanogaster, P. citrinum, X. laevis or A. nidulans.

In Vivo Alteration of Host Cell Glycosylation Using a Combinatorial DNALibrary

Certain methods of the invention are preferably (but need notnecessarily be) carried out using one or more nucleic acid libraries. Anexemplary feature of a combinatorial nucleic acid library of theinvention is that it comprises sequences encoding cellular targetingsignal peptides and sequences encoding proteins to be targeted (e.g.,enzymes or catalytic domains thereof, including but not limited to thosewhich mediate glycosylation).

In one embodiment, a combinatorial nucleic acid library comprises: (a)at least two nucleic acid sequences encoding different cellulartargeting signal peptides; and (b) at least one nucleic acid sequenceencoding a polypeptide to be targeted. In another embodiment, acombinatorial nucleic acid library comprises: (a) at least one nucleicacid sequence encoding a cellular targeting signal peptide; and (b) atleast two nucleic acid sequences encoding a polypeptide to be targetedinto a host cell. As described further below, a nucleic acid sequencederived from (a) and a nucleic acid sequence derived from (b) areligated to produce one or more fusion constructs encoding a cellulartargeting signal peptide functionally linked to a polypeptide domain ofinterest. One example of a functional linkage is when the cellulartargeting signal peptide is ligated to the polypeptide domain ofinterest in the same translational reading frame (“in-frame”).

In a preferred embodiment, a combinatorial DNA library expresses one ormore fusion proteins comprising cellular targeting signal peptidesligated in-frame to catalytic enzyme domains. The encoded fusion proteinpreferably comprises a catalytic domain of an enzyme involved inmammalian- or human-like modification of N-glycans. In a more preferredembodiment, the catalytic domain is derived from an enzyme selected fromthe group consisting of mannosidases, glycosyltransferases and otherglycosidases which is ligated in-frame to one or more targeting signalpeptides. The enzyme domain may be exogenous and/or endogenous to thehost cell. A particularly preferred signal peptide is one normallyassociated with a protein that undergoes ER to Golgi transport.

The combinatorial DNA library of the present invention may be used forproducing and localizing in vivo enzymes involved in mammalian- orhuman-like N-glycan modification. The fusion constructs of thecombinatorial DNA library are engineered so that the encoded enzymes arelocalized in the ER, Golgi or the trans-Golgi network of the host cellwhere they are involved in producing particular N-glycans on aglycoprotein of interest. Localization of N-glycan modifying enzymes ofthe present invention is achieved through an anchoring mechanism orthrough protein-protein interaction where the localization peptideconstructed from the combinatorial DNA library localizes to a desiredorganelle of the secretory pathway such as the ER, Golgi or the transGolgi network.

An example of a useful N-glycan, which is produced efficiently and insufficient quantities for further modification by human-like (complex)glycosylation reactions is Man₅GlcNAc₂. A sufficient amount ofMan₅GlcNAc₂ is needed on a glycoprotein of interest for furtherhuman-like processing in vivo (e.g., more than 30 mole %). TheMan₅GlcNAc₂ intermediate may be used as a substrate for further N-glycanmodification to produce GlcNAcMan₅GlcNAc₂ (FIG. 1B; see above).Accordingly, the combinatorial DNA library of the present invention maybe used to produce enzymes which subsequently produce GlcNAcMan₅GlcNAc₂,or other desired complex N-glycans, in a useful quantity.

A further aspect of the fusion constructs produced using thecombinatorial DNA library of the present invention is that they enablesufficient and often near complete intracellular N-glycan trimmingactivity in the engineered host cell. Preferred fusion constructsproduced by the combinatorial DNA library of the invention encode aglycosylation enzyme, e.g., a mannosidase, which is effectivelylocalized to an intracellular host cell compartment and thereby exhibitsvery little and preferably no extracellular activity. The preferredfusion constructs of the present invention that encode a mannosidaseenzyme are shown to localize where the N-glycans are modified, namely,the ER and the Golgi. The fusion enzymes of the present invention aretargeted to such particular organelles in the secretory pathway wherethey localize and act upon N-glycans such as Man₈GlcNAc₂ to produceMan₅GlcNAc₂ on a glycoprotein of interest.

Enzymes produced by the combinatorial DNA library of the presentinvention can modify N-glycans on a glycoprotein of interest as shownfor K3 or IFN-β proteins expressed in P. pastoris, as shown in FIG. 5and FIG. 6, respectively (see also Examples 2 and 11). It is, however,appreciated that other types of glycoproteins, without limitation,including erythropoietin, cytokines such as interferon-α, interferon-β,interferon-γ, interferon-ω, and granulocyte-CSF, coagulation factorssuch as factor VIII, factor IX, and human protein C, soluble IgEreceptor α-chain, IgG, IgG fragments, IgM, urokinase, chymase, and ureatrypsin inhibitor, IGF-binding protein, epidermal growth factor, growthhormone-releasing factor, annexin V fusion protein, angiostatin,vascular endothelial growth factor-2, myeloid progenitor inhibitoryfactor-1, osteoprotegerin, α-1 antitrypsin, DNase II and α-feto proteinsmay be glycosylated in this way.

Constructing a Combinatorial DNA Library of Fusion Constructs:

A combinatorial DNA library of fusion constructs features one or morecellular targeting signal peptides (“targeting peptides”) generallyderived from N-terminal domains of native proteins (e.g., by makingC-terminal deletions). Some targeting peptides, however, are derivedfrom the C-terminus of native proteins (e.g. SEC12). Membrane-boundproteins of the ER or the Golgi are preferably used as a source fortargeting peptide sequences. These proteins have sequences encoding acytosolic tail (ct), a transmembrane domain (tmd) and a stem region (sr)which are varied in length. These regions are recognizable by proteinsequence alignments and comparisons with known homologs and/or otherlocalized proteins (e.g., comparing hydrophobicity plots).

The targeting peptides are indicated herein as short (s), medium (m) andlong (l) relative to the parts of a type II membrane. The targetingpeptide sequence indicated as short (s) corresponds to the transmembranedomain (tmd) of the membrane-bound protein. The targeting peptidesequence indicated as long (l) corresponds to the length of thetransmembrane domain (tmd) and the stem region (sr). The targetingpeptide sequence indicated as medium (m) corresponds to thetransmembrane domain (tmd) and approximately half the length of the stemregion (sr). The catalytic domain regions are indicated herein by thenumber of nucleotide deletion with respect to its wild-typeglycosylation enzyme.

Sub-Libraries

In some cases a combinatorial nucleic acid library of the invention maybe assembled directly from existing or wild-type genes. In a preferredembodiment, the DNA library is assembled from the fusion of two or moresub-libraries. By the in-frame ligation of the sub-libraries, it ispossible to create a large number of novel genetic constructs encodinguseful targeted protein domains such as those which have glycosylationactivities.

Catalytic Domain Sub-Libraries Encoding Glycosylation Activities

One useful sub-library includes DNA sequences encoding enzymes such asglycosidases (e.g., mannosidases), glycosyltransferases (e.g.,fucosyltransferases, galactosyltransferases, glucosyltransferases),GlcNAc transferases and sialyltransferases. Catalytic domains may beselected from the host to be engineered, as well as from other relatedor unrelated organisms. Mammalian, plant, insect, reptile, algal orfungal enzymes are all useful and should be chosen to represent a broadspectrum of biochemical properties with respect to temperature and pHoptima. In a preferred embodiment, genes are truncated to give fragmentssome of which encode the catalytic domains of the enzymes. By removingendogenous targeting sequences, the enzymes may then be redirected andexpressed in other cellular loci.

The choice of such catalytic domains may be guided by the knowledge ofthe particular environment in which the catalytic domain is subsequentlyto be active. For example, if a particular glycosylation enzyme is to beactive in the late Golgi, and all known enzymes of the host organism inthe late Golgi have a certain pH optimum, or the late Golgi is known tohave a particular pH, then a catalytic domain is chosen which exhibitsadequate, and preferably maximum, activity at that pH, as discussedabove.

Targeting Peptide Sequence Sub-Libraries

Another useful sub-library includes nucleic acid sequences encodingtargeting signal peptides that result in localization of a protein to aparticular location within the ER, Golgi, or trans Golgi network. Thesetargeting peptides may be selected from the host organism to beengineered as well as from other related or unrelated organisms.Generally such sequences fall into three categories: (1) N-terminalsequences encoding a cytosolic tail (ct), a transmembrane domain (tmd)and part or all of a stem region (sr), which together or individuallyanchor proteins to the inner (lumenal) membrane of the Golgi; (2)retrieval signals which are generally found at the C-terminus such asthe HDEL (SEQ ID NO:5) or KDEL (SEQ ID NO:6) tetrapeptide; and (3)membrane spanning regions from various proteins, e.g., nucleotide sugartransporters, which are known to localize in the Golgi.

In the first case, where the targeting peptide consists of variouselements (ct, tmd and sr), the library is designed such that the ct, thetmd and various parts of the stem region are represented. Accordingly, apreferred embodiment of the sub-library of targeting peptide sequencesincludes ct, tmd, and/or sr sequences from membrane-bound proteins ofthe ER or Golgi. In some cases it may be desirable to provide thesub-library with varying lengths of sr sequence. This may beaccomplished by PCR using primers that bind to the 5′ end of the DNAencoding the cytosolic region and employing a series of opposing primersthat bind to various parts of the stem region.

Still other useful sources of targeting peptide sequences includeretrieval signal peptides, e.g. the tetrapeptides HDEL (SEQ ID NO:5) orKDEL (SEQ ID NO:6), which are typically found at the C-terminus ofproteins that are transported retrograde into the ER or Golgi. Stillother sources of targeting peptide sequences include (a) type IImembrane proteins, (b) the enzymes listed in Table 3, (c) membranespanning nucleotide sugar transporters that are localized in the Golgi,and (d) sequences referenced in Table 5 (The HDEL signal in column 1,cell 8 is shown in (SEQ ID NO:5).

TABLE 5 Sources of useful compartmental targeting sequences Gene orLocation of Sequence Organism Function Gene Product MNSI A. nidulansα-1,2-mannosidase ER MNSI A. niger α-1,2-mannosidase ER MNSI S.cerevisiae α-1,2-mannosidase ER GLSI S. cerevisiae glucosidase ER GLSIA. niger glucosidase ER GLSI A. nidulans glucosidase ER HDEL Universalin retrieval signal ER at C-terminus fungi SEC12 S. cerevisiae COPIIvesicle protein ER/Golgi SEC12 A. niger COPII vesicle protein ER/GolgiOCH1 S. cerevisiae 1,6-mannosyltransferase Golgi (cis) OCH1 P. pastoris1,6-mannosyltransferase Golgi (cis) MNN9 S. cerevisiae1,6-mannosyltransferase Golgi complex MNN9 A. niger undetermined GolgiVAN1 S. cerevisiae undetermined Golgi VAN1 A. niger undetermined GolgiANP1 S. cerevisiae undetermined Golgi HOCI S. cerevisiae undeterminedGolgi MNN10 S. cerevisiae undetermined Golgi MNN10 A. niger undeterminedGolgi MNN11 S. cerevisiae undetermined Golgi (cis) MNN11 A. nigerundetermined Golgi (cis) MNT1 S. cerevisiae 1,2-mannosyltransferaseGolgi (cis, medial KTR1 P. pastoris undetermined Golgi (medial) KRE2 P.pastoris undetermined Golgi (medial) KTR3 P. pastoris undetermined Golgi(medial) MNN2 S. cerevisiae 1,2-mannosyltransferase Golgi (medial) KTR1S. cerevisiae undetermined Golgi (medial) KTR2 S. cerevisiaeundetermined Golgi (medial) MNN1 S. cerevisiae 1,3-mannosyltransferaseGolgi (trans) MNN6 S. cerevisiae Phosphomannosyl- Golgi (trans)transferase 2,6 ST H. sapiens 2,6-sialyltransferase trans Golgi networkUDP-Gal T S. pombe UDP-Gal transporter Golgi

In any case, it is highly preferred that targeting peptide sequences areselected which are appropriate for the particular enzymatic activity oractivities to function optimally within the sequence of desiredglycosylation reactions. For example, in developing a modifiedmicroorganism capable of terminal sialylation of nascent N-glycans, aprocess which occurs in the late Golgi in humans, it is desirable toutilize a sub-library of targeting peptide sequences derived from lateGolgi proteins. Similarly, the trimming of Man₈GlcNAc₂ by anα-1,2-mannosidase to give Man₅GlcNAc₂ is an early step in complexN-glycan formation in humans (FIG. 1B). It is therefore desirable tohave this reaction occur in the ER or early Golgi of an engineered hostmicroorganism. A sub-library encoding ER and early Golgi retentionsignals is used.

A series of fusion protein constructs (i.e., a combinatorial DNAlibrary) is then constructed by functionally linking one or a series oftargeting peptide sequences to one or a series of sequences encodingcatalytic domains. In a preferred embodiment, this is accomplished bythe in-frame ligation of a sub-library comprising DNA encoding targetingpeptide sequences (above) with a sub-library comprising DNA encodingglycosylation enzymes or catalytically active fragments thereof (seebelow).

The resulting library comprises synthetic genes encoding targetingpeptide sequence-containing fusion proteins. In some cases it isdesirable to provide a targeting peptide sequence at the N-terminus of afusion protein, or in other cases at the C-terminus. In some cases,targeting peptide sequences may be inserted within the open readingframe of an enzyme, provided the protein structure of individual foldeddomains is not disrupted. Each type of fusion protein is constructed (ina step-wise directed or semi-random fashion) and optimal constructs maybe selected upon transformation of host cells and characterization ofglycosylation patterns in transformed cells using methods of theinvention.

Generating Additional Sequence Diversity

The method of this embodiment is most effective when a nucleic acid,e.g., a DNA library transformed into the host contains a large diversityof sequences, thereby increasing the probability that at least onetransformant will exhibit the desired phenotype. Single amino acidmutations, for example, may drastically alter the activity ofglycoprotein processing enzymes (Romero et al., 2000). Accordingly,prior to transformation, a DNA library or a constituent sub-library maybe subjected to one or more techniques to generate additional sequencediversity. For example, one or more rounds of gene shuffling, errorprone PCR, in vitro mutagenesis or other methods for generating sequencediversity, may be performed to obtain a larger diversity of sequenceswithin the pool of fusion constructs.

Expression Control Sequences

In addition to the open reading frame sequences described above, it isgenerally preferable to provide each library construct with expressioncontrol sequences, such as promoters, transcription terminators,enhancers, ribosome binding sites, and other functional sequences as maybe necessary to ensure effective transcription and translation of thefusion proteins upon transformation of fusion constructs into the hostorganism.

Suitable vector components, e.g., selectable markers, expression controlsequences (e.g., promoter, enhancers, terminators and the like) and,optionally, sequences required for autonomous replication in a hostcell, are selected as a function of which particular host cell ischosen. Selection criteria for suitable vector components for use in aparticular mammalian or a lower eukaryotic host cell are routine.Preferred lower eukaryotic host cells of the invention include Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp. Fusariumgramineum, Fusarium venenatum and Neurospora crassa. Where the host isPichia pastoris, suitable promoters include, for example, the AOX1,AOX2, GAPDH and P40 promoters.

Selectable Markers

It is also preferable to provide each construct with at least oneselectable marker, such as a gene to impart drug resistance or tocomplement a host metabolic lesion. The presence of the marker is usefulin the subsequent selection of transformants; for example, in yeast theURA3, HIS4, SUC2, G418, BLA, or SH BLE genes may be used. A multitude ofselectable markers are known and available for use in yeast, fungi,plant, insect, mammalian and other eukaryotic host cells.

Transformation

The nucleic acid library is then transformed into the host organism. Inyeast, any convenient method of DNA transfer may be used, such aselectroporation, the lithium chloride method, or the spheroplast method.In filamentous fungi and plant cells, conventional methods includeparticle bombardment, electroporation and agrobacterium mediatedtransformation. To produce a stable strain suitable for high-densityculture (e.g., fermentation in yeast), it is desirable to integrate theDNA library constructs into the host chromosome. In a preferredembodiment, integration occurs via homologous recombination, usingtechniques well-known in the art. For example, DNA library elements areprovided with flanking sequences homologous to sequences of the hostorganism. In this manner, integration occurs at a defined site in thehost genome, without disruption of desirable or essential genes.

In an especially preferred embodiment, library DNA is integrated intothe site of an undesired gene in a host chromosome, effecting thedisruption or deletion of the gene. For example, integration into thesites of the OCH1, MNN1, or MNN4 genes allows the expression of thedesired library DNA while preventing the expression of enzymes involvedin yeast hypermannosylation of glycoproteins. In other embodiments,library DNA may be introduced into the host via a nucleic acid molecule,plasmid, vector (e.g., viral or retroviral vector), chromosome, and maybe introduced as an autonomous nucleic acid molecule or by homologous orrandom integration into the host genome. In any case, it is generallydesirable to include with each library DNA construct at least oneselectable marker gene to allow ready selection of host organisms thathave been stably transformed. Recyclable marker genes such as ura3,which can be selected for or against, are especially suitable.

Screening and Selection Processes

After transformation of the host strain with the DNA library,transformants displaying a desired glycosylation phenotype are selected.Selection may be performed in a single step or by a series of phenotypicenrichment and/or depletion steps using any of a variety of assays ordetection methods. Phenotypic characterization may be carried outmanually or using automated high-throughput screening equipment.Commonly, a host microorganism displays protein N-glycans on the cellsurface, where various glycoproteins are localized.

One may screen for those cells that have the highest concentration ofterminal GlcNAc on the cell surface, for example, or for those cellswhich secrete the protein with the highest terminal GlcNAc content. Sucha screen may be based on a visual method, like a staining procedure, theability to bind specific terminal GlcNAc binding antibodies or lectinsconjugated to a marker (such lectins are available from E.Y.Laboratories Inc., San Mateo, Calif.), the reduced ability of specificlectins to bind to terminal mannose residues, the ability to incorporatea radioactively labeled sugar in vitro, altered binding to dyes orcharged surfaces, or may be accomplished by using a FluorescenceAssisted Cell Sorting (FACS) device in conjunction with a fluorophorelabeled lectin or antibody (Guillen, 1998).

Accordingly, intact cells may be screened for a desired glycosylationphenotype by exposing the cells to a lectin or antibody that bindsspecifically to the desired N-glycan. A wide variety ofoligosaccharide-specific lectins are available commercially (e.g., fromEY Laboratories, San Mateo, Calif.). Alternatively, antibodies tospecific human or animal N-glycans are available commercially or may beproduced using standard techniques. An appropriate lectin or antibodymay be conjugated to a reporter molecule, such as a chromophore,fluorophore, radioisotope, or an enzyme having a chromogenic substrate(Guillen et al., 1998. Proc. Natl. Acad. Sci. USA 95(14): 7888-7892)).

Screening may then be performed using analytical methods such asspectrophotometry, fluorimetry, fluorescence activated cell sorting, orscintillation counting. In other cases, it may be necessary to analyzeisolated glycoproteins or N-glycans from transformed cells. Proteinisolation may be carried out by techniques known in the art. In apreferred embodiment, a reporter protein is secreted into the medium andpurified by affinity chromatography (e.g. Ni-affinity orglutathione—S-transferase affinity chromatography). In cases where anisolated N-glycan is preferred, an enzyme such asendo-β-N-acetylglucosarnimidase (Genzyme Co., Boston, Mass.; New EnglandBiolabs, Beverly, Mass.) may be used to cleave the N-glycans fromglycoproteins. Isolated proteins or N-glycans may then be analyzed byliquid chromatography (e.g. HPLC), mass spectroscopy, or other suitablemeans. U.S. Pat. No. 5,595,900 teaches several methods by which cellswith desired extracellular carbohydrate structures may be identified. Ina preferred embodiment, MALDI-TOF mass spectrometry is used to analyzethe cleaved N-glycans.

Prior to selection of a desired transformant, it may be desirable todeplete the transformed population of cells having undesired phenotypes.For example, when the method is used to engineer a functionalmannosidase activity into cells, the desired transformants will havelower levels of mannose in cellular glycoprotein. Exposing thetransformed population to a lethal radioisotope of mannose in the mediumdepletes the population of transformants having the undesired phenotype,i.e. high levels of incorporated mannose (Huffaker T C and Robbins P W.,Proc Natl Acad Sci USA. 1983 December; 80(24):7466-70). Alternatively, acytotoxic lectin or antibody, directed against an undesirable N-glycan,may be used to deplete a transformed population of undesired phenotypes(e.g., Stanley P and Siminovitch L. Somatic Cell Genet. 1977 July;3(4):391-405). U.S. Pat. No. 5,595,900 teaches several methods by whichcells with a desired extracellular carbohydrate structures may beidentified. Repeatedly carrying out this strategy allows for thesequential engineering of more and more complex glycans in lowereukaryotes.

To detect host cells having on their surface a high degree of thehuman-like N-glycan intermediate GlcNAcMan₃GlcNAc₂, for example, one mayselect for transformants that allow for the most efficient transfer ofGlcNAc by GlcNAc Transferase from UDP-GlcNAc in an in vitro cell assay.This screen may be carried out by growing cells harboring thetransformed library under selective pressure on an agar plate andtransferring individual colonies into a 96-well microtiter plate. Aftergrowing the cells, the cells are centrifuged, the cells resuspended inbuffer, and after addition of UDP-GlcNAc and GnT II, the release of UDPis determined either by HPLC or an enzyme linked assay for UDP.Alternatively, one may use radioactively labeled UDP-GlcNAc and GnT II,wash the cells and then look for the release of radioactive GlcNAc byN-actylglucosaminidase. All this may be carried manually or automatedthrough the use of high throughput screening equipment. Transformantsthat release more UDP, in the first assay, or more radioactively labeledGlcNAc in the second assay, are expected to have a higher degree ofGlcNAcMan₃GlcNAc₂ on their surface and thus constitute the desiredphenotype. Similar assays may be adapted to look at the N-glycans onsecreted proteins as well.

Alternatively, one may use any other suitable screen such as a lectinbinding assay that is able to reveal altered glycosylation patterns onthe surface of transformed cells. In this case the reduced binding oflectins specific to terminal mannoses may be a suitable selection tool.Galantus nivalis lectin binds specifically to terminal α-1,3 mannose,which is expected to be reduced if sufficient mannosidase II activity ispresent in the Golgi. One may also enrich for desired transformants bycarrying out a chromatographic separation step that allows for theremoval of cells containing a high terminal mannose content. Thisseparation step would be carried out with a lectin column thatspecifically binds cells with a high terminal mannose content (e.g.,Galantus nivalis lectin bound to agarose, Sigma, St. Louis, Mo.) overthose that have a low terminal mannose content.

In addition, one may directly create such fusion protein constructs, asadditional information on the localization of active carbohydratemodifying enzymes in different lower eukaryotic hosts becomes availablein the scientific literature. For example, it is known that humanβ1,4-GalTr can be fused to the membrane domain of MNT, amannosyltransferase from S. cerevisiae, and localized to the Golgiapparatus while retaining its catalytic activity (Schwientek et al.,1995). If S. cerevisiae or a related organism is the host to beengineered one may directly incorporate such findings into the overallstrategy to obtain complex N-glycans from such a host. Several such genefragments in P. pastoris have been identified that are related toglycosyltransferases in S. cerevisiae and thus could be used for thatpurpose.

Alteration of Host Cell Glycosylation Using Fusion Constructs fromCombinatorial Libraries

The construction of a preferred combinatorial DNA library is illustratedschematically in FIG. 2 and described in Example 11. The fusionconstruct may be operably linked to a multitude of vectors, such asexpression vectors well-known in the art. A wide variety of such fusionconstructs were assembled using representative activities as shown inTable 6. Combinations of targeting peptide/catalytic domains may beassembled for use in targeting mannosidase, glycosyltransferase andglycosidase activities in the ER, Golgi and the trans Golgi networkaccording to the invention. Surprisingly, the same catalytic domain mayhave no effect to a very profound effect on N-glycosylation patterns,depending on the type of targeting peptide used (see, e.g., Table 7,Example 11).

Mannosidase Fusion Constructs

A representative example of a mannosidase fusion construct derived froma combinatorial DNA library of the invention is pFB8, which a truncatedSaccharomyces SEC12(m) targeting peptide (988-1296 nucleotides of SEC12from SwissProt P11655) ligated in-frame to a 187 N-terminal amino aciddeletion of a mouse α-mannosidase IA (Genbank AN 6678787). Thenomenclature used herein, thus, refers to the targetingpeptide/catalytic domain region of a glycosylation enzyme asSaccharomyces SEC12 (m)/mouse mannosidase IA Δ187. The encoded fusionprotein localizes in the ER by means of the SEC12 targeting peptidesequence while retaining its mannosidase catalytic domain activity andis capable of producing in vivo N-glycans having a Man₅GlcNAc₂ structure(Example 11; FIG. 6F, FIG. 7B).

The fusion construct pGC5, Saccharomyces MNS1(m)/mouse mannosidase IBΔ99, is another example of a fusion construct having intracellularmannosidase trimming activity (Example 11; FIG. 5D, FIG. 8B). Fusionconstruct pBC18-5 (Saccharomyces VAN1(s)/C. elegans mannosidase IB Δ80)is yet another example of an efficient fusion construct capable ofproducing N-glycans having a Man₅GlcNAc₂ structure in vivo. By creatinga combinatorial DNA library of these and other such mannosidase fusionconstructs according to the invention, a skilled artisan may distinguishand select those constructs having optimal intracellular trimmingactivity from those having relatively low or no activity. Methods usingcombinatorial DNA libraries of the invention are advantageous becauseonly a select few mannosidase fusion constructs may produce aparticularly desired N-glycan in vivo.

In addition, mannosidase trimming activity may be specific to aparticular protein of interest. Thus, it is to be further understoodthat not all targeting peptide/mannosidase catalytic domain fusionconstructs may function equally well to produce the proper glycosylationon a glycoprotein of interest. Accordingly, a protein of interest may beintroduced into a host cell transfected with a combinatorial DNA libraryto identify one or more fusion constructs which express a mannosidaseactivity optimal for the protein of interest. One skilled in the artwill be able to produce and select optimal fusion construct(s) using thecombinatorial DNA library approach described herein.

It is apparent, moreover, that other such fusion constructs exhibitinglocalized active mannosidase catalytic domains (or more generally,domains of any enzyme) may be made using techniques such as thoseexemplified in Example 11 and described herein. It will be a matter ofroutine experimentation for one skilled in the art to make and use thecombinatorial DNA library of the present invention to optimize, forexample, Man₅GlcNAc₂ production from a library of fusion constructs in aparticular expression vector introduced into a particular host cell.

Glycosyltransferase Fusion Constructs

Similarly, a glycosyltransferase combinatorial DNA library was madeusing the methods of the invention. A combinatorial DNA library ofsequences derived from glycosyltransferase I (GnTI) activities wereassembled with targeting peptides and screened for efficient productionin a lower eukaryotic host cell of a GlcNAcMan₅GlcNAc₂ N-glycanstructure on a marker glycoprotein. A fusion construct shown to produceGlcNAcMan₅GlcNAc₂ (pPB104), Saccharomyces MNN9(s)/human GnTI Δ38 wasidentified (Example 15). A wide variety of such GnTI fusion constructswere assembled (Example 15, Table 10). Other combinations of targetingpeptide/GnTI catalytic domains can readily be assembled by making acombinatorial DNA library. It is also apparent to one skilled in the artthat other such fusion constructs exhibiting glycosyltransferaseactivity may be made as demonstrated in Example 15. It will be a matterof routine experimentation for one skilled in the art to use thecombinatorial DNA library method described herein to optimizeGlcNAcMan₅GlcNAc₂ production using a selected fusion construct in aparticular expression vector and host cell line.

As stated above for mannosidase fusion constructs, not all targetingpeptide/GnTI catalytic domain fusion constructs will function equallywell to produce the proper glycosylation on a glycoprotein of interestas described herein. However, one skilled in the art will be able toproduce and select optimal fusion construct(s) using a DNA libraryapproach as described herein. Example 15 illustrates a preferredembodiment of a combinatorial DNA library comprising targeting peptidesand GnTI catalytic domain fusion constructs involved in producingglycoproteins with predominantly GlcNAcMan₅GlcNAc₂ structure.

Using Multiple Fusion Constructs to Alter Host Cell Glycosylation

In another example of using the methods and libraries of the inventionto alter host cell glycosylation, a P. pastoris strain with an OCH1deletion that expresses a reporter protein (K3) was transformed withmultiple fusion constructs isolated from combinatorial libraries of theinvention to convert high mannose N-glycans to human-like N-glycans(Example 15). First, the mannosidase fusion construct pFB8(Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) was transformed intoa P. pastoris strain lacking 1,6 initiating mannosyltransferasesactivity (i.e. och1 deletion; Example 1). Second, pPB103 comprising a K.lactis MNN2-2 gene (Genbank AN AF106080) encoding an UDP-GlcNActransporter was constructed to increase further production ofGlcNAcMan₅GlcNAc₂. The addition of the UDP-GlcNAc transporter increasedproduction of GlcNAcMan₅GlcNAc₂ significantly in the P. pastoris strainas illustrated in FIG. 10B. Third, pPB104 comprising Saccharomyces MNN9(s)/human GnTI Δ38 was introduced into the strain. This P. pastorisstrain is referred to as “PBP-3.”

It is understood by one skilled in the art that host cells such as theabove-described yeast strains can be sequentially transformed and/orco-transformed with one or more expression vectors. It is alsounderstood that the order of transformation is not particularly relevantin producing the glycoprotein of interest. The skilled artisanrecognizes the routine modifications of the procedures disclosed hereinmay provide improved results in the production of the glycoprotein ofinterest.

The importance of using a particular targeting peptide sequence with aparticular catalytic domain sequence becomes readily apparent from theexperiments described herein. The combinatorial DNA library provides atool for constructing enzyme fusions that are involved in modifyingN-glycans on a glycoprotein of interest, which is especially useful inproducing human-like glycoproteins. (Any enzyme fusion, however, may beselected using libraries and methods of the invention.) Desiredtransformants expressing appropriately targeted, activeα-1,2-mannosidase produce K3 with N-glycans of the structure Man₅GlcNAc₂as shown in FIGS. 5D and 5E. This confers a reduced molecular mass tothe cleaved glycan compared to the K3 of the parent OCH1 deletionstrain, as was detected by MALDI-TOF mass spectrometry in FIG. 5C.

Similarly, the same approach was used to produce another secretedglycoprotein: IFN-β comprising predominantly Man₅GlcNAc₂. TheMan₅GlcNAc₂ was removed by PNGase digestion (Papac et al. 1998Glycobiology 8, 445-454) and subjected to MALDI-TOF as shown in FIG.6A-6F. A single prominent peak at 1254 (m/z) confirms Man₅GlcNA₂production on IFN-β in FIG. 6E (pGC5) (Saccharomyces MNS1(m)/mousemannosidase IB Δ99) and 6F (pFB8) (Saccharomyces SEC12 (m)/mousemannosidase IA Δ187). Furthermore, in the P. pastoris strain PBP-3comprising pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187),pPB104 (Saccharomyces MNN9 (s)/human GnTI Δ38) and pPB103 (K. lactisMNN2-2 gene), the hybrid N-glycan GlcNAcMan₅GlcNAc₂ [b] was detected byMALDI-TOF (FIG. 10).

After identifying transformants with a high degree of mannose trimming,additional experiments were performed to confirm that mannosidase(trimming) activity occurred in vivo and was not predominantly theresult of extracellular activity in the growth medium (Example 13; FIGS.7-9).

Host Cells

Although the present invention is exemplified using a P. pastoris hostorganism, it is understood by those skilled in the art that othereukaryotic host cells, including other species of yeast and fungalhosts, may be altered as described herein to produce human-likeglycoproteins. The techniques described herein for identification anddisruption of undesirable host cell glycosylation genes, e.g. OCH1, isunderstood to be applicable for these and/or other homologous orfunctionally related genes in other eukaryotic host cells such as otheryeast and fungal strains. As described in Example 16, och1 mnn1 geneswere deleted from K. lactis to engineer a host cell leading to N-glycansthat are completely converted to Man₅GlcNAc₂ by 1,2-mannosidase (FIG.12C).

The MNN1 gene was cloned from K. lactis as described in Example 16. Thenucleic acid and deduced amino acid sequences of the K. lactis MNN1 geneare shown in SEQ ID NOS: 43 and 44, respectively. Using gene-specificprimers, a construct was made to delete the MNN1 gene from the genome ofK. lactis (Example 16). Host cells depleted in och1 and mnn1 activitiesproduce N-glycans having a Man₉GlcNAc₂ carbohydrate structure (see,e.g., FIG. 10). Such host cells may be engineered further using, e.g.,methods and libraries of the invention, to produce mammalian- orhuman-like glycoproteins.

Thus, in another embodiment, the invention provides an isolated nucleicacid molecule having a nucleic acid sequence comprising or consisting ofat least forty-five, preferably at least 50, more preferably at least 60and most preferably 75 or more nucleotide residues of the K. lactis MNN1gene (SEQ ID NO: 43), and homologs, variants and derivatives thereof.The invention also provides nucleic acid molecules that hybridize understringent conditions to the above-described nucleic acid molecules.Similarly, isolated polypeptides (including muteins, allelic variants,fragments, derivatives, and analogs) encoded by the nucleic acidmolecules of the invention are provided. In addition, also provided arevectors, including expression vectors, which comprise a nucleic acidmolecule of the invention, as described further herein. Similarly hostcells transformed with the nucleic acid molecules or vectors of theinvention are provided.

Another aspect of the present invention thus relates to a non-humaneukaryotic host strain expressing glycoproteins comprising modifiedN-glycans that resemble those made by human-cells. Performing themethods of the invention in species other than yeast and fungal cells isthus contemplated and encompassed by this invention. It is contemplatedthat a combinatorial nucleic acid library of the present invention maybe used to select constructs that modify the glycosylation pathway inany eukaryotic host cell system. For example, the combinatoriallibraries of the invention may also be used in plants, algae andinsects, and in other eukaryotic host cells, including mammalian andhuman cells, to localize proteins, including glycosylation enzymes orcatalytic domains thereof, in a desired location along a host cellsecretory pathway. Preferably, glycosylation enzymes or catalyticdomains and the like are targeted to a subcellular location along thehost cell secretory pathway where they are capable of functioning, andpreferably, where they are designed or selected to function mostefficiently.

As described in Examples 17 and 18, plant and insect cells may beengineered to alter the glycosylation of expressed proteins using thecombinatorial library and methods of the invention. Furthermore,glycosylation in mammalian cells, including human cells, may also bemodified using the combinatorial library and methods of the invention.It may be possible, for example, to optimize a particular enzymaticactivity or to otherwise modify the relative proportions of variousN-glycans made in a mammalian host cell using the combinatorial libraryand methods of the invention.

Examples of modifications to glycosylation which can be affected using amethod according to this embodiment of the invention are: (1)engineering a eukaryotic host cell to trim mannose residues fromMan₈GlcNAc₂ to yield a Man₅GlcNAc₂ N-glycan; (2) engineering eukaryotichost cell to add an N-acetylglucosamine (GlcNAc) residue to Man₅GlcNAc₂by action of GlcNAc transferase I; (3) engineering a eukaryotic hostcell to functionally express an enzyme such as an N-acetylglucosaminylTransferase (GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI), mannosidase II,fucosyltransferase (FT), galactosyl tranferase (GalT) or asialyltransferase (ST).

By repeating the method, increasingly complex glycosylation pathways canbe engineered into a target host, such as a lower eukaryoticmicroorganism. In one preferred embodiment, the host organism istransformed two or more times with DNA libraries including sequencesencoding glycosylation activities. Selection of desired phenotypes maybe performed after each round of transformation or alternatively afterseveral transformations have occurred. Complex glycosylation pathwayscan be rapidly engineered in this manner.

Sequential Glycosylation Reactions

In a preferred embodiment, such targeting peptide/catalytic domainlibraries are designed to incorporate existing information on thesequential nature of glycosylation reactions in higher eukaryotes.Reactions known to occur early in the course of glycoprotein processingrequire the targeting of enzymes that catalyze such reactions to anearly part of the Golgi or the ER. For example, the trimming ofMan₈GlcNAc₂ to Man₅GlcNAc₂ by mannosidases is an early step in complexN-glycan formation. Because protein processing is initiated in the ERand then proceeds through the early, medial and late Golgi, it isdesirable to have this reaction occur in the ER or early Golgi. Whendesigning a library for mannosidase I localization, for example, onethus attempts to match ER and early Golgi targeting signals with thecatalytic domain of mannosidase I.

Integration Sites

As one ultimate goal of this genetic engineering effort is a robustprotein production strain that is able to perform well in an industrialfermentation process, the integration of multiple genes into the host(e.g., fungal) chromosome preferably involves careful planning. Theengineered strain may likely have to be transformed with a range ofdifferent genes, and these genes will have to be transformed in a stablefashion to ensure that the desired activity is maintained throughout thefermentation process. As described herein, any combination of variousdesired enzyme activities may be engineered into the fungal proteinexpression host, e.g., sialyltransferases, mannosidases,fucosyltransferases, galactosyltransferases, glucosyltransferases,GlcNAc transferases, ER and Golgi specific transporters (e.g. syn andantiport transporters for UDP-galactose and other precursors), otherenzymes involved in the processing of oligosaccharides, and enzymesinvolved in the synthesis of activated oligosaccharide precursors suchas UDP-galactose, CMP-N-acetylneuraminic acid. Examples of preferredmethods for modifying glycosylation in a lower eukaryotic host cell,such as Pichia pastoris, are shown in Table 6. (The HDEL and KDEL signalpeptides in the second row of the third column are shown in SEQ ID NOS5and 6, respectively).

TABLE 6 Some preferred embodiments for modifying glycosylation in alower eukaroytic microorganism Suitable Suitable Suitable SourcesSuitable Transporters Catalytic of Localization Gene and/or StructureDesired Activities Sequences Deletions Phosphatases Man₅GlcNAc₂ α-1,2-Mns1 (N-terminus, OCH1 none mannosidase S. cerevisiae) MNN4 (murine,Och1 (N-terminus, MNN6 human, S. cerevisiae, Bacillus sp., P. pastoris)A. nidulans) Ktr1 Mnn9 Mnt1 (S. cerevisiae) KDEL, HDEL (C-terminus)GlcNAcMan₅GlcNAc₂ GlcNAc Och1 (N-terminus, OCH1 UDP-GlcNAc TransferaseI, S. cerevisiae, MNN4 transporter (human, P. pastoris) MNN6 (human,murine, rat KTR1 (N-terminus) murine, etc.) Mnn1 (N-terminus, K. lactis)S. cerevisiae) UDPase Mnt1 (N-terminus, (human) S. cerevisiae) GDPase(N-terminus, S. cerevisiae) GlcNAcMan₃GlcNAc₂ mannosidase Ktr1 OCH1UDP-GlcNAc II Mnn1 (N-terminus, MNN4 transporter S. cerevisiae) MNN6(human, Mnt1 (N-terminus, murine, S. cerevisiae) K. lactis) Kre2/Mnt1UDPase (S. cerevisiae) (human) Kre2 (P. pastoris) Ktr1 (S. cerevisiae)Ktr1 (P. pastoris) Mnn1 (S. cerevisiae) GlcNAc⁽²⁻⁴⁾Man₃GlcNAc₂ GlcNAcMnn1 (N-terminus, OCH1 UDP-GlcNAc Transferase S. cerevisiae) MNN4transporter II, III, IV, V Mnt1 (N-terminus, MNN6 (human, (human, S.cerevisiae) murine, murine) Kre2/Mnt1 K. lactis) (S. cerevisiae) UDPaseKre2 (P. pastoris) (human) Ktr1 (S. cerevisiae) Ktr1 (P. pastoris) Mnn1(S. cerevisiae) Gal⁽¹⁻⁴⁾GlcNAc⁽²⁻⁴⁾Man₃GlcNAc₂ β-1,4- Mnn1 (N-terminus,OCH1 UDP- Galactosyl S. cerevisiae) MNN4 Galactose transferaseMnt1(N-terminus, MNN6 transporter (human) S. cerevisiae) (human,Kre2/Mnt1 S. pombe) (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S.cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae)NANA⁽¹⁻⁴Gal⁽¹⁻⁴⁾GlcNAc⁽²⁻⁴⁾⁾Man₃GlcNAc₂ α-2,6- KTR1 OCH1 CMP-Sialyltransferase MNN1 (N-terminus, MNN4 Sialic acid (human) S.cerevisiae) MNN6 transporter α-2,3- MNT1 (N-terminus, (human)Sialyltransferase S. cerevisiae) Kre2/Mnt1 (S. cerevisiae) Kre2 (P.pastoris) Ktr1 (S. cerevisiae) Ktr1 (P. pastoris) MNN1 (S. cerevisiae)

As any strategy to engineer the formation of complex N-glycans into ahost cell such as a lower eukaryote involves both the elimination aswell as the addition of particular glycosyltransferase activities, acomprehensive scheme will attempt to coordinate both requirements. Genesthat encode enzymes that are undesirable serve as potential integrationsites for genes that are desirable. For example, 1,6 mannosyltransferaseactivity is a hallmark of glycosylation in many known lower eukaryotes.The gene encoding alpha-1,6 mannosyltransferase (OCH1) has been clonedfrom S. cerevisiae and mutations in the gene give rise to a viablephenotype with reduced mannosylation. The gene locus encoding alpha-1,6mannosyltransferase activity therefore is a prime target for theintegration of genes encoding glycosyltransferase activity. In a similarmanner, one can choose a range of other chromosomal integration sitesthat, based on a gene disruption event in that locus, are expected to:(1) improve the cells ability to glycosylate in a more human-likefashion, (2) improve the cells ability to secrete proteins, (3) reduceproteolysis of foreign proteins and (4) improve other characteristics ofthe process that facilitate purification or the fermentation processitself.

Target Glycoproteins

The methods described herein are useful for producing glycoproteins,especially glycoproteins used therapeutically in humans. Glycoproteinshaving specific glycoforms may be especially useful, for example, in thetargeting of therapeutic proteins. For example, mannose-6-phosphate hasbeen shown to direct proteins to the lysosome, which may be essentialfor the proper function of several enzymes related to lysosomal storagedisorders such as Gaucher's, Hunter's, Hurler's, Scheie's, Fabry's andTay-Sachs disease, to mention just a few. Likewise, the addition of oneor more sialic acid residues to a glycan side chain may increase thelifetime of a therapeutic glycoprotein in vivo after administration.Accordingly, host cells (e.g., lower eukaryotic or mammalian) may begenetically engineered to increase the extent of terminal sialic acid inglycoproteins expressed in the cells. Alternatively, sialic acid may beconjugated to the protein of interest in vitro prior to administrationusing a sialic acid transferase and an appropriate substrate. Changes ingrowth medium composition may be employed in addition to the expressionof enzyme activities involved in human-like glycosylation to produceglycoproteins more closely resembling human forms (S. Weikert, et al.,Nature Biotechnology, 1999, 17, 1116-1121; Werner, Noe, et al 1998Arzneimittelforschung 48(8): 870-880; Weikert, Papac et al., 1999;Andersen and Goochee 1994 Cur. Opin. Biotechnol. 5: 546-549; Yang andButler 2000 Biotechnol. Bioengin. 68(4): 370-380). Specific glycanmodifications to monoclonal antibodies (e.g. the addition of a bisectingGlcNAc) have been shown to improve antibody dependent cell cytotoxicity(Umana P., et al. 1999), which may be desirable for the production ofantibodies or other therapeutic proteins.

Therapeutic proteins are typically administered by injection, orally,pulmonary, or other means. Examples of suitable target glycoproteinswhich may be produced according to the invention include, withoutlimitation: erythropoietin, cytokines such as interferon-α,interferon-β, interferon-γ, interferon-ω, and granulocyte-CSF,coagulation factors such as factor VIII, factor IX, and human protein C,soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins,urokinase, chymase, and urea trypsin inhibitor, IGF-binding protein,epidermal growth factor, growth hormone-releasing factor, annexin Vfusion protein, angiostatin, vascular endothelial growth factor-2,myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1-antitrypsinand α-feto proteins.

The following are examples which illustrate the compositions and methodsof this invention. These examples should not be construed as limiting:the examples are included for the purposes of illustration only.

EXAMPLE 1 Cloning and Disruption of the OCH1 Gene in P. pastoris

Generation of an OCH1 Mutant of P. pastoris:

A 1215 bp ORF of the P. pastoris OCH1 gene encoding a putative α-1,6mannosyltransferase was amplified from P. pastoris genomic DNA (strainX-33, Invitrogen, Carlsbad, Calif.) using the oligonucleotides5′-ATGGCGAAGGCAGATGGCAGT-3′ (SEQ ID NO:7) and5′-TTAGTCCTTCCAACTTCCTTC-3′ (SEQ ID NO:8) which were designed based onthe P. pastoris OCH1 sequence (Japanese Patent Application PublicationNo. 8-336387). Subsequently, 2685 bp upstream and 1175 bp downstream ofthe ORF of the OCH1 gene were amplified from a P. pastoris genomic DNAlibrary (Boehm, T. et al. Yeast 1999 May; 15(7):563-72) using theinternal oligonucleotides 5′-ACTGCCATCTGCCTTCGCCAT-3′ (SEQ ID NO:9) inthe OCH1 gene, and 5′-GTAATACGACTCACTATAGGGC-3′ T7 (SEQ ID NO:10) and5′-AATTAACCCTCACTAAAGGG-3′ T3 (SEQ ID NO:11) oligonucleotides in thebackbone of the library bearing plasmid lambda ZAP II (Stratagene, LaJolla, Calif.). The resulting 5075 bp fragment was cloned into thepCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) and designated pBK9.

After assembling a gene knockout construct that substituted the OCH1reading frame with a HIS4 resistance gene, P. pastoris was transformedand colonies were screened for temperature sensitivity at 37° C. OCH1mutants of S. cerevisiae are temperature sensitive and are slow growersat elevated temperatures. One can thus identify functional homologs ofOCH1 in P. pastoris by complementing an OCH1 mutant of S. cerevisiaewith a P. pastoris DNA or cDNA library. About 20 temperature sensitivestrains were further subjected to a colony PCR screen to identifycolonies with a deleted och1 gene. Several och1 deletions were obtained.

The linearized pBK9.1, which has 2.1 kb upstream sequence and 1.5 kbdown stream sequence of OCH1 gene cassette carrying Pichia HIS4 gene,was transformed into P. pastoris BK1 [GS115 (his4 Invitrogen Corp., SanDiego, Calif.) carrying the human IFN-β gene in the AOX1 locus] to knockout the wild-type OCH1 gene. The initial screening of transformants wasperformed using histidine drop-out medium followed by replica plating toselect the temperature sensitive colonies. Twenty out of two hundredhistidine-positive colonies showed a temperature sensitive phenotype at37° C. To exclude random integration of pBK9.1 into the Pichia genome,the 20 temperature-sensitive isolates were subjected to colony PCR usingprimers specific to the upstream sequence of the integration site and toHIS4 ORF. Two out of twenty colonies were och1 defective and furtheranalyzed using a Southern blot and a Western blot indicating thefunctional och1 disruption by the och1 knock-out construct. Genomic DNAwere digested using two separate restriction enzymes BglII and ClaI toconfirm the och1 knock-out and to confirm integration at the openreading frame. The Western Blot showed och1 mutants lacking a discreteband produced in the GS 115 wild type at 46.2 kDa.

EXAMPLE 2 Engineering of P. pastoris with α-1,2-Mannosidase to ProduceMan₅GlcNAc₂-Containing IFN-β Precursors

An α-1,2-mannosidase is required for the trimming of Man₈GlcNAc₂ toyield Man₅GlcNAc₂, an essential intermediate for complex N-glycanformation. While the production of a Man₅GlcNAc₂ precursor is essential,it is not necessarily sufficient for the production of hybrid andcomplex glycans because the specific isomer of Man₅GlcNAc₂ may or maynot be a substrate for GnTI. An och1 mutant of P. pastoris is engineeredto express secreted human interferon-β under the control of an aoxpromoter. A DNA library is constructed by the in-frame ligation of thecatalytic domain of human mannosidase IB (an α-1,2-mannosidase) with asub-library including sequences encoding early Golgi and ER localizationpeptides. The DNA library is then transformed into the host organism,resulting in a genetically mixed population wherein individualtransformants each express interferon-β as well as a syntheticmannosidase gene from the library. Individual transformant colonies arecultured and the production of interferon is induced by addition ofmethanol. Under these conditions, over 90% of the secreted protein isglycosylated interferon-β.

Supernatants are purified to remove salts and low-molecular weightcontaminants by C₁₈ silica reversed-phase chromatography. Desiredtransformants expressing appropriately targeted, activeα-1,2-mannosidase produce interferon-β including N-glycans of thestructure Man₅GlcNAc₂, which has a reduced molecular mass compared tothe interferon-β of the parent strain. The purified interferon-β isanalyzed by MALDI-TOF mass spectroscopy and colonies expressing thedesired form of interferon-β are identified.

EXAMPLE 3 Generation of an Och1 Mutant Strain Expressing anα-1,2-Mannosidase, GnTI and GnTII for Production of a Human-LikeGlycoprotein

The 1215 bp open reading frame of the P. pastoris OCH1 gene as well as2685 bp upstream and 1175 bp downstream was amplified by PCR (see alsoWO 02/00879), cloned into the pCR2.1-TOPO vector (Invitrogen) anddesignated pBK9. To create an och1 knockout strain containing multipleauxotrophic markers, 100 μg of pJN329, a plasmid containing anoch1::URA3 mutant allele flanked with SfiI restriction sites wasdigested with SfiI and used to transform P. pastoris strain JC308(Cereghino et al. Gene 263 (2001) 159-169) by electroporation. Followingincubation on defined medium lacking uracil for 10 days at roomtemperature, 1000 colonies were picked and re-streaked. URA⁺ clones thatwere unable to grow at 37° C., but grew at room temperature, weresubjected to colony PCR to test for the correct integration of theoch1::URA3 mutant allele. One clone that exhibited the expected PCRpattern was designated YJN153. The Kringle 3 domain of human plasminogen(K3) was used as a model protein. A Neo^(R) marked plasmid containingthe K3 gene was transformed into strain YJN153 and a resulting strain,expressing K3, was named BK64-1.

Plasmid pPB103, containing the Kluyveromyces lactis MNN2-2 gene whichencodes a Golgi UDP-N-acetylglucosamine transporter was constructed bycloning a blunt BglII-HindIII fragment from vector pDL02 (Abeijon et al.(1996) Proc. Natl. Acad. Sci. U.S.A. 93:5963-5968) into BglII and BamHIdigested and blunt ended pBLADE-SX containing the P. pastoris ADE1 gene(Cereghino et al. (2001) Gene 263:159-169). This plasmid was linearizedwith EcoNI and transformed into strain BK64-1 by electroporation and onestrain confirmed to contain the MNN2-2 by PCR analysis was named PBP1.

A library of mannosidase constructs was generated, comprising in-framefusions of the leader domains of several type I or type II membraneproteins from S. cerevisiae and P. pastoris fused with the catalyticdomains of several α-1,2-mannosidase genes from human, mouse, fly, wormand yeast sources (see, e.g., WO02/00879, incorporated herein byreference). This library was created in a P. pastoris HIS4 integrationvector and screened by linearizing with SalI, transforming byelectroporation into strain PBP1, and analyzing the glycans releasedfrom the K3 reporter protein. One active construct chosen was a chimeraof the 988-1296 nucleotides (C-terminus) of the yeast SEC12 gene fusedwith a N-terminal deletion of the mouse α-1,2-mannosidase IA gene (FIG.3), which was missing the 187 nucleotides. A P. pastoris strainexpressing this construct was named PBP2.

A library of GnTI constructs was generated, comprising in-frame fusionsof the same leader library with the catalytic domains of GnTI genes fromhuman, worm, frog and fly sources (WO 02/00879). This library wascreated in a P. pastoris ARG4 integration vector and screened bylinearizing with AatII, transforming by electroporation into strainPBP2, and analyzing the glycans released from K3. One active constructchosen was a chimera of the first 120 bp of the S. cerevisiae MNN9 genefused to a deletion of the human GnTI gene, which was missing the first154 bp. A P. pastoris strain expressing this construct was named PBP3.

A library of GnTII constructs was generated, which comprised in-framefusions of the leader library with the catalytic domains of GnTII genesfrom human and rat sources (WO 02/00879). This library was created in aP. pastoris integration vector containing the NST^(R) gene conferringresistance to the drug nourseothricin. The library plasmids werelinearized with EcoRI, transformed into strain RDP27 by electroporation,and the resulting strains were screened by analysis of the releasedglycans from purified K3.

Materials for the Following Reactions

MOPS, sodium cacodylate, manganese chloride, UDP-galactose andCMP-N-acetylneuraminic acid were from Sigma. Trifluoroacetic acid (TFA)was from Sigma/Aldrich, Saint Louis, Mo. Recombinant ratα2,6-sialyltransferase from Spodoptera frugiperda andβ1,4-galactosyltransferase from bovine milk were from Calbiochem (SanDiego, Calif.). Protein N-glycosidase F, mannosidases, andoligosaccharides were from Glyko (San Rafael, Calif.). DEAE ToyoPearlresin was from TosoHaas. Metal chelating “HisBind” resin was fromNovagen (Madison, Wis.). 96-well lysate-clearing plates were fromPromega (Madison, Wis.). Protein-binding 96-well plates were fromMillipore (Bedford, Mass.). Salts and buffering agents were from Sigma(St. Louis, Mo.). MALDI matrices were from Aldrich (Milwaukee, Wis.).

Protein Purification

Kringle 3 was purified using a 96-well format on a Beckman BioMek 2000sample-handling robot (Beckman/Coulter Ranch Cucamonga, Calif.). Kringle3 was purified from expression media using a C-terminal hexa-histidinetag. The robotic purification is an adaptation of the protocol providedby Novagen for their HisBind resin. Briefly, a 150 uL (μL) settledvolume of resin is poured into the wells of a 96-well lysate-bindingplate, washed with 3 volumes of water and charged with 5 volumes of 50mM NiSO4 and washed with 3 volumes of binding buffer (5 mM imidazole,0.5M NaCl, 20 mM Tris-HCL pH7.9). The protein expression media isdiluted 3:2, media/PBS (60 mM PO₄, 16 mM KCl, 822 mM NaCl pH7.4) andloaded onto the columns. After draining, the columns are washed with 10volumes of binding buffer and 6 volumes of wash buffer (30 mM imidazole,0.5M NaCl, 20 mM Tris-HCl pH7.9) and the protein is eluted with 6volumes of elution buffer (1M imidazole, 0.5M NaCl, 20 mM Tris-HClpH7.9). The eluted glycoproteins are evaporated to dryness bylyophilyzation.

Release of N-Linked Glycans

The glycans are released and separated from the glycoproteins by amodification of a previously reported method (Papac, et al. A. J. S.(1998) Glycobiology 8, 445-454). The wells of a 96-well MultiScreen IP(Immobilon-P membrane) plate (Millipore) are wetted with 100 uL ofmethanol, washed with 3×150 uL of water and 50 uL of RCM buffer (8Murea, 360 mM Tris, 3.2 mM EDTA pH8.6), draining with gentle vacuum aftereach addition. The dried protein samples are dissolved in 30 uL of RCMbuffer and transferred to the wells containing 10 uL of RCM buffer. Thewells are drained and washed twice with RCM buffer. The proteins arereduced by addition of 60 uL of 0.1M DTT in RCM buffer for 1 hr at 37°C. The wells are washed three times with 300 uL of water andcarboxymethylated by addition of 60 uL of 0.1M iodoacetic acid for 30min in the dark at room temperature. The wells are again washed threetimes with water and the membranes blocked by the addition of 100 uL of1% PVP 360 in water for 1 hr at room temperature. The wells are drainedand washed three times with 300 uL of water and deglycosylated by theaddition of 30 uL of 10 mM NH₄HCO₃ pH 8.3 containing one milliunit ofN-glycanase (Glyko). After 16 hours at 37° C., the solution containingthe glycans was removed by centrifugation and evaporated to dryness.

Matrix Assisted Laser Desorption Ionization Time of Flight MassSpectrometry

Molecular weights of the glycans were determined using a Voyager DE PROlinear MALDI-TOF (Applied Biosciences) mass spectrometer using delayedextraction. The dried glycans from each well were dissolved in 15 uL ofwater and 0.5 uL spotted on stainless steel sample plates and mixed with0.5 uL of S-DHB matrix (9 mg/mL of dihydroxybenzoic acid, 1 mg/mL of5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) and allowedto dry.

Ions were generated by irradiation with a pulsed nitrogen laser (337 nm)with a 4 ns pulse time. The instrument was operated in the delayedextraction mode with a 125 ns delay and an accelerating voltage of 20kV. The grid voltage was 93.00%, guide wire voltage was 0.10%, theinternal pressure was less than 5×10-7 torr, and the low mass gate was875 Da. Spectra were generated from the sum of 100-200 laser pulses andacquired with a 2 GHz digitizer. Man₅GlcNAc₂ oligosaccharide was used asan external molecular weight standard. All spectra were generated withthe instrument in the positive ion mode. The estimated mass accuracy ofthe spectra was 0.5%.

EXAMPLE 4 Engineering a Strain to Produce Galactosyltransferase

Galactosyltransferase Reaction

Approximately 2 mg of protein (r-K3:hPg [PBP6-5]) was purified bynickel-affinity chromatography, extensively dialyzed against 0.1% TFA,and lyophilized to dryness. The protein was redissolved in 150 μL of 50mM MOPS, 20 mM MnCl2, pH7.4. After addition of 32.5 μg (533 nmol) ofUDP-galactose and 4 mU of β1,4-galactosyltransferase, the sample wasincubated at 37° C. for 18 hours. The samples were then dialyzed against0.1% TFA for analysis by MALDI-TOF mass spectrometry.

The spectrum of the protein reacted with galactosyltransferase showed anincrease in mass consistent with the addition of two galactose moietieswhen compared with the spectrum of a similar protein sample incubatedwithout enzyme. Protein samples were next reduced, carboxymethylated anddeglycosylated with PNGase F. The recovered N-glycans were analyzed byMALDI-TOF mass spectrometry. The mass of the predominant glycan from thegalactosyltransferase reacted protein was greater than that of thecontrol glycan by a mass consistent with the addition of two galactosemoieties (325.4 Da).

EXAMPLE 5 Engineering a Strain to Express Functional and ActiveMannosidase II

To generate a human-like glycoform, a microorganism is engineered toexpress a mannosidase II enzyme which removes the two remaining terminalmannoses from the structure GlcNAcMan₅GlcNAc₂ (see FIG. 1B). A DNAlibrary including sequences encoding cis and medial Golgi localizationsignals is fused in-frame to a library encoding mannosidase II catalyticdomains. The host organism is a strain, e.g. a yeast, that is deficientin hypermannosylation (e.g. an och1 mutant) and provides N-glycanshaving the structure GlcNAcMan₅GlcNAc₂ in the Golgi and/or ER. Aftertransformation, organisms having the desired glycosylation phenotype areselected. An in vitro assay is used in one method. The desired structureGlcNAcMan₃GlcNAc₂ (but not the undesired GlcNAcMan₅GlcNAc₂) is asubstrate for the enzyme GlcNAc Transferase II (see FIG. 1B).Accordingly, single colonies may be assayed using this enzyme in vitroin the presence of the substrate, UDP-GlcNAc. The release of UDP isdetermined either by HPLC or an enzymatic assay for UDP. Alternatively,radioactively labeled UDP-GlcNAc or MALDI-TOF may be used.

The foregoing in vitro assays are conveniently performed on individualcolonies using high-throughput screening equipment. Alternatively alectin binding assay is used. In this case the reduced binding oflectins specific for terminal mannoses allows the selection oftransformants having the desired phenotype. For example, Galantusnivalis lectin binds specifically to terminal α-1,3-mannose, theconcentration of which is reduced in the presence of operativelyexpressed mannosidase II activity. In one suitable method, G. nivalislectin attached to a solid agarose support (available from SigmaChemical, St. Louis, Mo.) is used to deplete the transformed populationof cells having high levels of terminal α-1,3-mannose.

EXAMPLE 6 Engineering a Strain to Express Sialyltransferase

The enzymes α-2,3-sialyltransferase and α2,6-sialyltransferase addterminal sialic acid to galactose residues in nascent human N-glycans,leading to mature glycoproteins (see “α2,3 ST; α2,6 ST” in FIG. 1B). Inhuman cells, the reactions occur in the trans Golgi or TGN. Accordingly,a DNA library is constructed by the in-frame fusion of sequencesencoding sialyltransferase catalytic domains with sequences encodingtrans Golgi or TGN localization signals (Malissard et al. BiochemBiophys Res Commun 2000 Jan. 7; 267(1):169-73; Borsig et al. BiochemBiophys Res Commun 1995 May 5; 210(1):14-20). The host organism is astrain, e.g. a yeast, that is deficient in hypermannosylation (e.g., anoch1 mutant), which provides N-glycans having terminal galactoseresidues in the late Golgi or TGN, and provides a sufficientconcentration of CMP-sialic acid in the late Golgi or TGN. Followingtransformation, transformants having the desired phenotype are selected,e.g., using a fluorescent antibody specific for N-glycans having aterminal sialic acid. In addition, the strains are engineered to producethe CMP-NANA precursors.

Sialyltransferase Reaction

After resuspending the (galactosyltransferase reacted) (Example 4)proteins in 10 μL of 50 mM sodium cacodylate buffer pH6.0, 300 μg (488nmol) of CMP-N-acetylneuraminic acid (CMP-NANA) dissolved in 15 μL ofthe same buffer, and 5 μL (2 mU) of recombinant α-2,6 sialyltransferasewere added. After incubation at 37° C. for 15 hours, an additional 200μg of CMP-NANA and 1 mU of sialyltransferase were added. The proteinsamples were incubated for an additional 8 hours and then dialyzed andanalyzed by MALDI-TOF-MS as above. The spectrum of the glycoproteinreacted with sialyltransferase showed an increase in mass when comparedwith that of the starting material (the protein aftergalactosyltransferase reaction). The N-glycans were released andanalyzed as above. The increase in mass of the two ion-adducts of thepredominant glycan was consistent with the addition of two sialic acidresidues (580 and 583 Da).

EXAMPLE 7 Engineering a Strain to Express UDP-GlcNAc Transporter

The cDNA of human Golgi UDP-GlcNAc transporter has been cloned by Ishidaand coworkers. (Ishida, N., et al. 1999 J. Biochem. 126(1): 68-77).Guillen and coworkers have cloned the canine kidney Golgi UDP-GlcNActransporter by phenotypic correction of a Kluyveromyces lactis mutantdeficient in Golgi UDP-GlcNAc transport. (Guillen, E., et al. 1998).Thus a mammalian Golgi UDP-GlcNAc transporter gene has all of thenecessary information for the protein to be expressed and targetedfunctionally to the Golgi apparatus of yeast. These or other clonedtransporter genes may be engineered into a host organism to provideUDP-GlcNAc substrates for efficient GnT reactions in the Golgi and/or ERof the host. FIG. 10B demonstrates the effect of a strain expressing aK. lactis UDP-GlcNAc transporter. In comparison to FIG. 10A, which lacksa UDP-GlcNAc transporter, the effect of adding a UDP-GlcNAc transportershows a dramatic increase in the production of GlcNAcMan₅GlcNAc₂.

EXAMPLE 8 Engineering a Strain to Express GDP-Fucose Transporter

The rat liver Golgi membrane GDP-fucose transporter has been identifiedand purified by Puglielli, L. and C. B. Hirschberg 1999 J. Biol. Chem.274(50):35596-35600. The corresponding gene can be identified usingstandard techniques, such as N-terminal sequencing and Southern blottingusing a degenerate DNA probe. The intact gene is then expressed in ahost microorganism that also expresses a fucosyltransferase.

EXAMPLE 9 Engineering a Strain to Express UDP-Galactose Transporter

Human UDP-galactose (UDP-Gal) transporter has been cloned and shown tobe active in S. cerevisiae. (Kainuma, M., et al. 1999 Glycobiology 9(2):133-141). A second human UDP-galactose transporter (hUGT1) has beencloned and functionally expressed in Chinese Hamster Ovary Cells. Aoki,K., et al. 1999 J. Biochem. 126(5): 940-950. Likewise, Segawa andcoworkers have cloned a UDP-galactose transporter fromSchizosaccharomyces pombe (Segawa, H., et al. 1999 Febs Letters 451(3):295-298). These or other sequences encoding UDP-galactose transporteractivities may be introduced into a host cell directly or may be used asa component of a sub-library of the invention to engineer a strainhaving increased UDP-galactose transporter activity.

EXAMPLE 10 Engineering a Strain to Express CMP-Sialic Acid Transporter

Human CMP-sialic acid transporter (hCST) has been cloned and expressedin Lec 8 CHO cells by Aoki and coworkers (1999). Molecular cloning ofthe hamster CMP-sialic acid transporter has also been achieved (Eckhardtand Gerardy Schahn 1997 Eur. J. Biochem. 248(1): 187-192). Thefunctional expression of the murine CMP-sialic acid transporter wasachieved in Saccharomyces cerevisiae by Berninsone, P., et al. 1997 J.Biol. Chem. 272 (19):12616-12619. These or other sequences encodingCMP-sialic acid transporter activities may be introduced into a hostcell directly or may be used as a component of a sub-library of theinvention to engineer a strain having increased CMP-sialic acidtransporter activity.

EXAMPLE 11 Engineering of P. pastoris to Produce Man₅GlcNA₂ as thePredominant N-Glycan Structure Using a Combinatorial DNA Library

An och1 mutant of P. pastoris (see Examples 1 and 3) was engineered toexpress and secrete proteins such as the kringle 3 domain of humanplasminogen (K3) under the control of the inducible AOXI promoter. TheKringle 3 domain of human plasminogen (K3) was used as a model protein.A DNA fragment encoding the K3 was amplified using Pfu turbo polymerase(Strategene, La Jolla, Calif.) and cloned into EcoRI and XbaI sites ofpPICZαA (Invitrogen, Carlsbad, Calif.), resulting in a C-terminal 6-Histag. In order to improve the N-linked glycosylation efficiency of K3(Hayes et al. 1975 J. Arch. Biochem. Biophys. 171, 651-655), Pro₄₆ wasreplaced with Ser₄₆ using site-directed mutagenesis. The resultingplasmid was designated pBK64. The correct sequence of the PCR constructwas confirmed by DNA sequencing.

A combinatorial DNA library was constructed by the in-frame ligation ofmurine α-1,2-mannosidase IB (Genbank AN 6678787) and IA (Genbank AN6754619) catalytic domains with a sub-library including sequencesencoding Cop II vesicle, ER, and early Golgi localization peptidesaccording to Table 6. The combined DNA library was used to generateindividual fusion constructs, which were then transformed into the K3expressing host organism, resulting in a genetically mixed populationwherein individual transformants each express K3 as well as alocalization signal/mannosidase fusion gene from the library. Individualtransformants were cultured and the production of K3 was induced bytransfer to a methanol containing medium. Under these conditions, after24 hours of induction, over 90% of the protein in the medium was K3. TheK3 reporter protein was purified from the supernatant to remove saltsand low-molecular weight contaminants by Ni-affinity chromatography.Following affinity purification, the protein was desalted by sizeexclusion chromatography on a Sephadex G10 resin (Sigma, St. Louis, Mo.)and either directly subjected to MALDI-TOF analysis described below orthe N-glycans were removed by PNGase digestion as described below(Release of N-glycans) and subjected to MALDI-TOF analysis Miele et al.1997 Biotechnol. Appl. Biochem. 25, 151-157.

Following this approach, a diverse set of transformants were obtained;some showed no modification of the N-glycans compared to the och1knockout strain; and others showed a high degree of mannose trimming(FIGS. 5D, 5E). Desired transformants expressing appropriately targeted,active α-1,2-mannosidase produced K3 with N-glycans of the structureMan₅GlcNAc₂. This confers a reduced molecular mass to the glycoproteincompared to the K3 of the parent och1 deletion strain, a differencewhich was readily detected by MALDI-TOF mass spectrometry (FIG. 5).Table 7 indicates the relative Man₅GlcNAc₂ production levels.

TABLE 7 A representative combinatorial DNA library of localizationsequences/catalytic domains exhibiting relative levels of Man₅GlcNAc₂production. Targeting peptide sequences MNS1(s) MNS1(m) MNS1(l) SEC12(s)SEC12(m) Catalytic Mouse mannosidase FB4 FB5 FB6 FB7 FB8 Domains 1A Δ187++ + − ++ ++++ Mouse mannosidase GB4 GB5 GB6 GB7 GB8 1B Δ58 ++ + + ++ +Mouse mannosidase GC4 GC5 GC6 GC7 GC8 1B Δ99 − +++ + + + Mousemannosidase GD4 GD5 GD6 GD7 GD8 1B Δ170 − − − + +

TABLE 8 Another combinatorial DNA library of localizationsequences/catalytic domains exhibiting relative levels of Man₅GlcNAc₂production. Targeting peptide sequences VAN1(s) VAN1(m) VAN1(l) MNN10(s)MNN10(m) MNN10(l) Catalytic C. elegans BC18-5 BC19 BC20 BC27 BC28 BC29Domains mannosidase 1B +++++ ++++ +++ +++++ +++++ +++ Δ80 C. elegansBB18 BB19 BB20 BB18 BB19 BB20 mannosidase 1B +++++ +++++ ++++ ++++++++++ ++++ Δ31

Targeting peptides were selected from MNS I (SwissProt P32906) in S.cerevisiae (long, medium and short) (see supra Nucleic Acid Libraries;Combinatorial DNA Library of Fusion Constructs) and SEC12 (SwissProtP11655) in S. cerevisiae (988-1140 nucleotides: short) and (988-1296:medium). Although majority of the targeting peptide sequences wereN-terminal deletions, some targeting peptide sequences, such as SEC12were C-terminal deletions. Catalytic domains used in this experimentwere selected from mouse mannosidase 1A with a 187 amino acid N-terminaldeletion; and mouse mannosidase 1B with a 58, 99 and 170 amino aciddeletion. The number of (+)s, as used herein, indicates the relativelevels of Man₅GlcNA₂ production. The notation (−) indicates no apparentproduction of Man₅GlcNA₂. The notation (+) indicates less than 10%production of Man₅GlcNA₂ The notation (++) indicates about 10-20%production of Man₅GlcNA₂. The notation with (+++) indicates about 20-40%production of Man₅GlcNA₂. The notation with (++++) indicates about 50%production of Man₅GlcNA₂. The notation with (+++++) indicates greaterthan 50% production of Man₅GlcNA₂.

Table 9 shows relative amount of Man₅GlcNAc₂ on secreted K3. Six hundredand eight (608) different strains of P. pastoris, Δoch1 were generatedby transforming them with a single construct of a combinatorial geneticlibrary that was generated by fusing nineteen (19) α-1,2 mannosidasecatalytic domains to thirty-two (32) fungal ER, and cis-Golgi leaders.

TABLE 9 Amount of Man₅GlcNAc₂ Number of on secreted K3 constructs (% oftotal glycans) (%) N.D.* 19 (3.1)  0-10% 341 (56.1) 10-20% 50 (8.2)20-40&  75 (12.3) 40-60%  72 (11.8) More than 60%  51 (8.4) ^(†) Total608 (100)  *Several fusion constructs were not tested because thecorresponding plasmids could not be propagated in E.coli prior totransformation into P.pastoris. ^(†) Clones with the highest degree ofMan₅GlcNAc₂ trimming (30/51) were further analyzed for mannosidaseactivity in the supernatant of the medium. The majority (28/30)displayed detectable mannosidase activity in the supernatant (e.g. FIG.4B). Only two constructs displayed high Man₅GlcNAc₂ levels, whilelacking mannosidase activity in the medium (e.g. FIG. 4C).

Table 7 shows two constructs pFB8 and pGC5, among others, displayingMan₅GlcNA₂. Table 8 shows a more preferred construct, pBC18-5, a S.cerevisiae VAN1(s) targeting peptide sequence (from SwissProt 23642)ligated in-frame to a C. elegans mannosidase IB (Genbank AN CAA98114) 80amino acid N-terminal deletion (Saccharomyces Van1(s)/C. elegansmannosidase IB Δ80). This fusion construct also produces a predominantMan₅GlcNA₂ structure, as shown in FIG. 5E. This construct was shown toproduce greater than 50% Man₅GlcNA₂ (+++++).

Generation of a Combinatorial Localization/Mannosidase Library:

Generating a combinatorial DNA library of α-1,2-mannosidase catalyticdomains fused to targeting peptides required the amplification ofmannosidase domains with varying lengths of N-terminal deletions from anumber of organisms. To approach this goal, the full length open readingframes (ORFs) of α-1,2-mannosidases were PCR amplified from either cDNAor genomic DNA obtained from the following sources: Homo sapiens, Musmusculus, Drosophila melanogaster, Caenorhabditis elegans, Aspergillusnidulans and Penicillium citrinum. In each case, DNA was incubated inthe presence of oligonucleotide primers specific for the desiredmannosidase sequence in addition to reagents required to perform the PCRreaction. For example, to amplify the ORF of the M. musculusα-1,2-mannosidase IA, the 5′-primer ATGCCCGTGGGGGGCCTGTTGCCGCTCTTCAGTAGC(SEQ ID NO:12) and the 3′-primerTCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG (SEQ ID NO:13) were incubatedin the presence of Pfu DNA polymerase (Stratagene, La Jolla, Calif.) andamplified under the conditions recommended by Stratagene using thecycling parameters: 94° C. for 1 min (1 cycle); 94° C. for 30 sec, 68°C. for 30 sec, 72° C. for 3 min (30 cycles). Following amplification theDNA sequence encoding the ORF was incubated at 72° C. for 5 min with 1UTaq DNA polymerase (Promega, Madison, Wis.) prior to ligation intopCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) and transformed into TOP10chemically competent E. coli, as recommended by Invitrogen. The clonedPCR product was confirmed by ABI sequencing using primers specific forthe mannosidase ORF.

To generate the desired N-terminal truncations of each mannosidase, thecomplete ORF of each mannosidase was used as the template in asubsequent round of PCR reactions wherein the annealing position of the5′-primer was specific to the 5′-terminus of the desired truncation andthe 3′-primer remained specific for the original 3′-terminus of the ORF.To facilitate subcloning of the truncated mannosidase fragment into theyeast expression vector, pJN347 (FIG. 2C) AsaI and PacI restrictionsites were engineered onto each truncation product, at the 5′- and3′-termini respectively. The number and position of the N-terminaltruncations generated for each mannosidase ORF depended on the positionof the transmembrane (TM) region in relation to the catalytic domain(CD). For instance, if the stem region located between the TM and CD wasless than 150 bp, then only one truncation for that protein wasgenerated. If, however, the stem region was longer than 150 bp theneither one or two more truncations were generated depending on thelength of the stem region.

An example of how truncations for the M. musculus mannosidase IA(Genbank AN 6678787) were generated is described herein, with a similarapproach being used for the other mannosidases. FIG. 3 illustrates theORF of the M. musculus α-1,2-mannosidase IA with the predictedtransmembrane and catalytic domains being highlighted in bold. Based onthis structure, three 5′-primers were designed (annealing positionsunderlined in FIG. 3) to generate the Δ65-, Δ105- and Δ187-N-terminaldeletions. Using the Δ65 N-terminal deletion as an example the 5′-primerused was 5′-GGCGCGCCGACTCCTCCAAGCTGCTCAGCGGGGTCCTGTTCCAC-3′ (SEQ IDNO:14) (with the AscI restriction site highlighted in bold) inconjunction with the 3′-primer5′-CCTTAATTAATCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG-3′ (SEQ ID NO:15)(with the Pad restriction site highlighted in bold). Both of theseprimers were used to amplify a 1561 bp fragment under the conditionsoutlined above for amplifying the full length M. musculus mannosidase 1AORF. Furthermore, like the product obtained for the full length ORF, thetruncated product was also incubated with Taq DNA polymerase, ligatedinto pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.), transformed into TOP10and ABI sequenced. After having amplified and confirmed the sequence ofthe truncated mannosidase fragment, the resulting plasmid, pCR2.1-Δ65mMannIA, was digested with AscI and PacI in New England Biolabs buffer#4 (Beverly, Mass.) for 16 h at 37° C. In parallel, the pJN347 (FIG. 2C)was digested with the same enzymes and incubated as described above.Post-digestion, both the pJN347 (FIG. 2C) back-bone and the truncatedcatalytic domain were gel extracted and ligated using the Quick LigationKit (New England Biolabs, Beverly, Mass.), as recommended by themanufacturers, and transformed into chemically competent DH5α cells(Invitrogen, Carlsbad, Calif.). Colony PCR was used to confirm thegeneration of the pJN347-mouse Mannosidase IAΔ65 construct.

Having generated a library of truncated α-1,2-mannosidase catalyticdomains in the yeast expression vector pJN347 (FIG. 2C) the remainingstep in generating the targeting peptide/catalytic domain library was toclone in-frame the targeting peptide sequences (FIG. 2). Both thepJN347-mannosidase constructs (FIG. 2D) and the pCR2.1TOPO-targetingpeptide constructs (FIG. 2B) such as were incubated overnight at 37° C.in New England Biolabs buffer #4 in the presence of the restrictionenzymes NotI and AscI. Following digestion, both the pJN347-mannosidaseback-bone and the targeting peptide regions were gel-extracted andligated using the Quick Ligation Kit (New England Biolabs, Beverly,Mass.), as recommended by the manufacturers, and transformed intochemically competent DH5α cells (Invitrogen, Carlsbad, Calif.).Subsequently, the pJN347-targeting peptide/mannosidase constructs wereABI sequenced to confirm that the generated fusions were in-frame. Theestimated size of the final targeting peptide/alpha-1,2-mannosidaselibrary contains over 1300 constructs generated by the approachdescribed above. FIG. 2 illustrates construction of the combinatorialDNA library.

Engineering a P. pastoris OCH1 Knock-Out Strain with MultipleAuxotrophic Markers.

The first step in plasmid construction involved creating a set ofuniversal plasmids containing DNA regions of the KEX1 gene of P.pastoris (Boehm et al. Yeast 1999 May; 15(7):563-72) as space holdersfor the 5′ and 3′ regions of the genes to be knocked out. The plasmidsalso contained the S. cerevisiae Ura-blaster (Alani et al., Genetics116, 541-545. 1987) as a space holder for the auxotrophic markers, andan expression cassette with a multiple cloning site for insertion of aforeign gene. A 0.9-kb fragment of the P. pastoris KEX1-5′ region wasamplified by PCR using primersGGCGAGCTCGGCCTACCCGGCCAAGGCTGAGATCATTTGTCCAGCTTCA GA (SEQ ID NO:16) andGCCCACGTCGACGGATCCGTTTAAACATCGATTGGAGAGGCTGACACC GCTACTA (SEQ ID NO:17)and P. pastoris genomic DNA as a template and cloned into the SacI, SalIsites of pUC19 (New England Biolabs, Beverly, Mass.). The resultingplasmid was cut with BamHI and SalI, and a 0.8-kb fragment of theKEX1-3′ region that had been amplified using primersCGGGATCCACTAGTATTTAAATCATATGTGCGAGTGTACAACTCTTCCC ACATGG (SEQ ID NO:18)and GGACGCGTCGACGGCCTACCCGGCCGTACGAGGAATTTCTCGG ATGACTCTTTTC (SEQ IDNO:19) was cloned into the open sites creating pJN262. This plasmid wascut with BamHI and the 3.8-kb BamHI, BglII fragment of pNKY51 (Alani etal. 1987) was inserted in both possible orientations resulting inplasmids pJN263 (FIG. 4A) and pJN284 (FIG. 4B).

An expression cassette was created with NotI and PacI as cloning sites.The GAPDH promoter of P. pastoris was amplified using primersCGGGATCCCTCGAGAGATCTTTTTTGTAGAAATGTCTTGGTGCCT (SEQ ID NO:20) andGGACATGCATGCACTAGTGCGGCCGCCACGTGATAGTTGTTCA ATTGATTGAAATAGGGACAA (SEQ IDNO:21) and plasmid pGAPZ-A (Invitrogen) as template and cloned into theBamHI, SphI sites of pUC19 (New England Biolabs, Beverly, Mass.) (FIG.4B). The resulting plasmid was cut with SpeI and SphI and the CYC1transcriptional terminator region (“TT”) that had been amplified usingprimers CCTTGCTAGCTTAATTAACCGCGGCACGTCCGACGGCGGCCCA CGGGTCCCA (SEQ IDNO:22) and GGACATGCATGCGGATCCCTTAAGAGCCGGCAGCTTGCAAATTAAAGCCTTCGAGCGTCCC (SEQ ID NO:23) and plasmid pPICZ-A (Invitrogen) as atemplate was cloned into the open sites creating pJN261 (FIG. 4B).

A knockout plasmid for the P. pastoris OCH1 gene was created bydigesting pJN263 with SalI and SpeI and a 2.9-kb DNA fragment of theOCH1-5′ region, which had been amplified using the primersGAACCACGTCGACGGCCATTGCGGCCAAAACCTTTTTTCCTATT CAAACACAAGGCATTGC (SEQ IDNO:24) and CTCCAATACTAGTCGAAGATTATCTTCTACGGTGCCTGGACTC (SEQ ID NO:25)and P. pastoris genomic DNA as a template, was cloned into the opensites (FIG. 4C). The resulting plasmid was cut with EcoRI and PmeI and a1.0-kb DNA fragment of the OCH1-3′ region that had been generated usingthe primers TGGAAGGTTTAAACAAAGCTAGAGTAAAATAGATATAGCGAG ATTAGAGAATG (SEQID NO:26) and AAGAATTCGGCTGGAAGGCCTTGTACCTTGATGTAGTTCCCGTT TTCATC (SEQID NO:27) was inserted to generate pJN298 (FIG. 4C). To allow for thepossibility to simultaneously use the plasmid to introduce a new gene,the BamHI expression cassette of pJN261 (FIG. 4B) was cloned into theunique BamHI site of pJN298 (FIG. 4C) to create pJN299 (FIG. 4E).

The P. pastoris Ura3-blaster cassette was constructed using a similarstrategy as described in Lu. P., et al. 1998 (Cloning and disruption ofthe β-isopropylmalate dehydrogenase gene (Leu2) of Pichia stipidis withURA3 and recovery of the double auxotroph. Appl. Microbiol. Biotechnol.49, 141-146.) A 2.0-kb PstI, SpeI fragment of P. pastoris URA3 wasinserted into the PstI, XbaI sites of pUC19 (New England Biolabs,Beverly, Mass.) to create pJN306 (FIG. 4D). Then a 0.7-kb SacI, PvuIIDNA fragment of the lacZ open reading frame was cloned into the SacI,SmaI sites to yield pJN308 (FIG. 4D). Following digestion of pJN308(FIG. 4D) with PstI, and treatment with T4 DNA polymerase, theSacI-PvuII fragment from lacZ that had been blunt-ended with T4 DNApolymerase was inserted generating pJN315 (FIG. 4D). The lacZ/URA3cassette was released by digestion with SacI and SphI, blunt ended withT4 DNA polymerase and cloned into the backbone of pJN299 that had beendigested with PmeI and AflII and blunt ended with T4 DNA polymerase. Theresulting plasmid was named pJN329 (FIG. 4E).

A HIS4 marked expression plasmid was created by cutting pJN261 (FIG. 4F)with EcoICRI (FIG. 4F). A 2.7 kb fragment of the Pichia pastoris HIS4gene that had been amplified using the primersGCCCAAGCCGGCCTTAAGGGATCTCCTGATGACTGACTCACTGATAATA AAAATACGG (SEQ IDNO:28) and GGGCGCGTATTTAAATACTAGTGGATCTATCGAATCTAAATGTAAGTTA AAATCTCTAA(SEQ ID NO:29) cut with NgoMIV and SwaI and then blunt-ended using T4DNA polymerase, was then ligated into the open site. This plasmid wasnamed pJN337 (FIG. 4F). To construct a plasmid with a multiple cloningsite suitable for fusion library construction, pJN337 was cut with NocIand PacI and the two oligonucleotidesGGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT (SEQ ID NO:30) andTAAGGCGCGCCGAATTCATTTAAATCTGCAGGGC (SEQ ID NO:31), that had beenannealed in vitro were ligated into the open sites, creating pJN347(FIG. 4F).

To create an och1 knockout strain containing multiple auxotrophicmarkers, 100 μg of pJN329 was digested with SfiI and used to transformP. pastoris strain JC308 (Cereghino et al. Gene 263 (2001) 159-169) byelectroporation. Following transformation, the URA dropout plates wereincubated at room temperature for 10 days. One thousand (1000) colonieswere picked and restreaked. All 1000 clones were then streaked onto 2sets of URA dropout plates. One set was incubated at room temperature,whereas the second set was incubated at 37° C. The clones that wereunable to grow at 37° C., but grew at room temperature, were subjectedto colony PCR to test for the correct OCHI knockout. One clone thatshowed the expected PCR signal (about 4.5 kb) was designated YJN153.

EXAMPLE 12 Characterization of the Combinatorial DNA Library

Positive transformants screened by colony PCR confirming integration ofthe mannosidase construct into the P. pastoris genome were subsequentlygrown at room temperature in 50 ml BMGY buffered methanol-complex mediumconsisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphatebuffer, pH 6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, and 1%glycerol as a growth medium) until OD_(600nm) 2-6 at which point theywere washed with 10 ml BMMY (buffered methanol-complex medium consistingof 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, and 1.5% methanol as agrowth medium) media prior to induction of the reporter protein for 24hours at room temperature in 5 ml BMMY. Consequently, the reporterprotein was isolated and analyzed by mass spectrophotometry and HPLC tocharacterize its glycan structure. Using the targeting peptides in Table6, mannosidase catalytic domains localized to either the ER or the Golgishowed significant level of trimming of a glycan predominantlycontaining Man₈GlcNAc₂ to a glycan predominantly containing Man₅GlcNAc₂.This is evident when the glycan structure of the reporter glycoproteinis compared between that of P. pastoris och1 knock-out in FIGS. 5C, 6Cand the same strain transformed with M. musculus mannosidase constructsas shown in FIGS. 5D, 5E, 6D-6F. FIGS. 5 and 6 show expression ofconstructs generated from the combinatorial DNA library which showsignificant mannosidase activity in P. pastoris. Expression of pGC5(Saccharomyces MNS1(m)/mouse mannosidase IB Δ99) (FIGS. 5D, 6E) produceda protein which has approximately 30% of all glycans trimmed toMan₅GlcNAc₂, while expression of pFB8 (Saccharomyces SEC/2(m)/mousemannosidase IA Δ187) (FIG. 6F) produced approximately 50% Man₅GlcNAc₂and expression of pBC18-5 (Saccharomyces VAN1(s)/C. elegans mannosidaseIB Δ80) (FIG. 5E) produced 70% Man₅GlcNAc₂.

Release of N-Glycans

The glycans were released and separated from the glycoproteins by amodification of a previously reported method (Papac et al. 1998Glycobiology 8, 445-454). After the proteins were reduced andcarboxymethylated and the membranes blocked, the wells were washed threetime with water. The protein was deglycosylated by the addition of 30 μlof 10 mM NH₄HCO₃ pH 8.3 containing one milliunit of N-glycanase (Glyko,Novato, Calif.). After 16 hr at 37° C., the solution containing theglycans was removed by centrifugation and evaporated to dryness.

Matrix Assisted Laser Desorption Ionization Time of Flight MassSpectrometry

After the N-glycans were released by PNGase digestion, they wereanalyzed by Matrix Assisted Laser Desorption Ionization Time of FlightMass Spectrometry. Molecular weights of the glycans were determinedusing a Voyager DE PRO linear MALDI-TOF (Applied Biosciences) massspectrometer using delayed extraction. The dried glycans from each wellwere dissolved in 15 μl of water and 0.5 μl was spotted on stainlesssteel sample plates and mixed with 0.5 μl of S-DHB matrix (9 mg/ml ofdihydroxybenzoic acid, 1 mg/ml of 5-methoxysalicilic acid in 1:1water/acetonitrile 0.1% TFA) and allowed to dry. Ions were generated byirradiation with a pulsed nitrogen laser (337 nm) with a 4 ns pulsetime. The instrument was operated in the delayed extraction mode with a125 ns delay and an accelerating voltage of 20 kV. The grid voltage was93.00%, guide wire voltage was 0.1%, the internal pressure was less than5×10⁻⁷ ton, and the low mass gate was 875 Da. Spectra were generatedfrom the sum of 100-200 laser pulses and acquired with a 500 MHzdigitizer. Man₅GlcNAc₂ oligosaccharide was used as an external molecularweight standard. All spectra were generated with the instrument in thepositive ion mode.

EXAMPLE 13 Trimming In Vivo by Alpha-1,2-Mannosidase

To ensure that the novel engineered strains of Example 11 in factproduced the desired Man₅GlcNAc₂ structure in vivo, cell supernatantswere tested for mannosidase activity (see FIGS. 7-9). For eachconstruct/host strain described below, HPLC was performed at 30° C. witha 4.0 mm×250 mm column of Altech (Avondale, Pa., USA) Econosil-NH₂ resin(5 μm) at a flow rate of 1.0 ml/min for 40 min. In FIGS. 7 and 8,degradation of the standard Man₉GlcNAc₂ [b] was shown to occur resultingin a peak which correlates to Man₈GlcNAc₂. In FIG. 7, the Man₉GlcNAc₂[b] standard eluted at 24.61 min and Man₅GlcNAc₂ [a] eluted at 18.59min. In FIG. 8, Man₉GlcNAc₂ eluted at 21.37 min and Man₅GlcNAc₂ at 15.67min. In FIG. 9, the standard Man₈GlcNAc₂ [b] was shown to elute at 20.88min.

P. pastoris cells comprising plasmid pFB8 (Saccharomyces SEC12 (m)/mousemannosidase IA Δ187) were grown at 30° C. in BMGY to an OD600 of about10. Cells were harvested by centrifugation and transferred to BMMY toinduce the production of K3 (kringle 3 from human plasminogen) undercontrol of an AOX1 promoter. After 24 hours of induction, cells wereremoved by centrifugation to yield an essentially clear supernatant. Analiquot of the supernatant was removed for mannosidase assays and theremainder was used for the recovery of secreted soluble K3. A singlepurification step using CM-sepharose chromatography and an elutiongradient of 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted ina 95% pure K3 eluting between 300-500 mM NaCl. N-glycan analysis of theK3 derived glycans is shown in FIG. 6F. The earlier removed aliquot ofthe supernatant was further tested for the presence of secretedmannosidase activity. A commercially available standard of2-aminobenzamide-labeled N-linked-type oligomannose 9 (Man9-2-AB)(Glyko, Novato, Calif.) was added to: BMMY (FIG. 7A), the supernatantfrom the above aliquot (FIG. 7B), and BMMY containing 10 ng of 75 mU/mLof α-1,2-mannosidase from Trichoderma reesei (obtained from Contreras etal., WO 02/00856 A2) (FIG. 7C). After incubation for 24 hours at roomtemperature, samples were analyzed by amino silica HPLC to determine theextent of mannosidase trimming.

P. pastoris cells comprising plasmid pGC5 (Saccharomyces MNS1(m)/mousemannosidase IB ΔA99) were similarly grown and assayed. Cells were grownat room temperature in BMGY to an OD600 of about 10. Cells wereharvested by centrifugation and transferred to BMMY to induce theproduction of K3 under control of an AOX1 promoter. After 24 hours ofinduction, cells were removed by centrifugation to yield an essentiallyclear supernatant. An aliquot of the supernatant was removed formannosidase assays and the remainder was used for the recovery ofsecreted soluble K3. A single purification step using CM-sepharosechromatography and an elution gradient of 25 mM NaAc, pH5.0 to 25 mMNaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500mM NaCl. N-glycan analysis of the K3 derived glycans is shown in FIG.5D. The earlier removed aliquot of the supernatant was further testedfor the presence of secreted mannosidase activity as shown in FIG. 8B. Acommercially available standard of Man9-2-AB (Glyko, Novato, Calif.)were added to: BMMY (FIG. 8A), supernatant from the above aliquot (FIG.8B), and BMMY containing 10 ng of 75 mU/mL of α-1,2-mannosidase fromTrichoderma reesei (obtained from Contreras et al., WO 02/00856 A2)(FIG. 8C). After incubation for 24 hours at room temperature, sampleswere analyzed by amino silica HPLC to determine the extent ofmannosidase trimming.

Man9-2-AB was used as a substrate and it is evident that after 24 hoursof incubation, mannosidase activity was virtually absent in thesupernatant of the pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IAA187) strain digest (FIG. 7B) and pGC5 (Saccharomyces MNS1(m)/mousemannosidase IB Δ99) strain digest (FIG. 8B) whereas the positive control(purified α-1,2-mannosidase from T. reesei obtained from Contreras)leads to complete conversion of Man₉GlcNAc₂ to Man₅GlcNAc₂ under thesame conditions, as shown in FIGS. 7C and 8C. This is conclusive datashowing in vivo mannosidase trimming in P. pastoris pGC5 strain; andpFB8 strain, which is distinctly different from what has been reportedto date (Contreras et al., WO 02/00856 A2).

FIG. 9 further substantiates localization and activity of themannosidase enzyme. P. pastoris comprising pBC18-5 (SaccharomycesVAN1(s)/C. elegans mannosidase IB Δ80) was grown at room temperature inBMGY to an OD600 of about 10. Cells were harvested by centrifugation andtransferred to BMMY to induce the production of K3 under control of anAOX1 promoter. After 24 hours of induction, cells were removed bycentrifugation to yield an essentially clear supernatant. An aliquot ofthe supernatant was removed for mannosidase assays and the remainder wasused for the recovery of secreted soluble K3. A single purification stepusing CM-sepharose chromatography and an elution gradient 25 mM NaAc,pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 elutingbetween 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans isshown in FIG. 5E. The earlier removed aliquot of the supernatant wasfurther tested for the presence of secreted mannosidase activity asshown in FIG. 9B. A commercially available standard of Man8-2-AB (Glyko,Novato, Calif.) was added to: BMMY (FIG. 9A), supernatant from the abovealiquot pBC18-5 (Saccharomyces VAN1(s)/C. elegans mannosidase IB Δ80)(FIG. 9B), and BMMY containing media from a different fusion constructpDD28-3 (Saccharomyces MNN10(m) (from SwissProt 50108)/H. sapiensmannosidase IB Δ99) (FIG. 9C). After incubation for 24 hours at roomtemperature, samples were analyzed by amino silica HPLC to determine theextent of mannosidase trimming. FIG. 9B demonstrates intracellularmannosidase activity in comparison to a fusion construct pDD28-3(Saccharomyces MNN10(m) H. sapiens mannosidase IB Δ99) exhibiting anegative result (FIG. 9C).

EXAMPLE 14 pH Optimum Assay of Engineered α-1,2-Mannosidase

P. pastoris cells comprising plasmid pBB27-2 (Saccharomyces MNN10 (s)(from SwissProt 50108)/C. elegans mannosidase IB Δ31) were grown at roomtemperature in BMGY to an OD600 of about 17. About 80 μL of these cellswere inoculated into 600 μL BMGY and were grown overnight. Subsequently,cells were harvested by centrifugation and transferred to BMMY to inducethe production of K3 (kringle 3 from human plasminogen) under control ofan AOX1 promoter. After 24 hours of induction, cells were removed bycentrifugation to yield an essentially clear supernatant (pH 6.43). Thesupernatant was removed for mannosidase pH optimum assays.Fluorescence-labeled Man₈GlcNAc₂ (0.5 μg) was added to 20 μL ofsupernatant adjusted to various pH (FIG. 11) and incubated for 8 hoursat room temperature. Following incubation the sample was analyzed byHPLC using an Econosil NH2 4.6×250 mm, 5 micron bead, amino-bound silicacolumn (Altech, Avondale, Pa.). The flow rate was 1.0 ml/min for 40 minand the column was maintained to 30° C. After eluting isocratically (68%A:32% B) for 3 min, a linear solvent gradient (68% A:32% B to 40% A:60%B) was employed over 27 min to elute the glycans (18). Solvent A(acetonitrile) and solvent B (ammonium formate, 50 mM, pH 4.5. Thecolumn was equilibrated with solvent (68% A:32% B) for 20 mM betweenruns.

EXAMPLE 15 Engineering of P. pastoris to Produce N-Glycans with theStructure GlcNAcMan₅GlcNAc₂

GlcNAc Transferase I activity is required for the maturation of complexand hybrid N-glycans (U.S. Pat. No. 5,834,251). Man₅GlcNAc₂ may only betrimmed by mannosidase II, a necessary step in the formation of humanglycoforms, after the addition of N-acetylglucosamine to the terminalα-1,3 mannose residue of the trimannose stem by GlcNAc Transferase I(Schachter, 1991 Glycobiology 1(5):453-461). Accordingly, acombinatorial DNA library was prepared including DNA fragments encodingsuitably targeted catalytic domains of GlcNAc Transferase I genes fromC. elegans and Homo sapiens; and localization sequences from GLS, MNS,SEC, MNN9, VAN1, ANP1, HOC1, MNN10, MNN11, MNT1, KTR1, KTR2, MNN2, MNN5,YUR1, MNN1, and MNN6 from S. cerevisiae and P. pastoris putativeα-1,2-mannosyltransferases based on the homology from S. cerevisiae: D2,D9 and J3, which are KTR homologs. Table 10 includes but does not limittargeting peptide sequences such as SEC and OCH1, from P. pastoris andK. lactis GnTI, (See Table 6 and Table 10)

TABLE 10 A representative combinatorial library of targeting peptidesequences/ catalytic domain for UDP-N-Acetylglucosaminyl Transferase I(GnTI) Targeting peptide OCHI(s) OCHI(m) OCHI(l) MNN9(s) MNN9(m)Catalytic Human, GnTI, Δ38 PB105 PB106 PB107 PB104 N/A Domain Human,GnTI, Δ86 NB12 NB13 NB14 NB15 NB C. elegans, GnTI, Δ88 OA12 OA13 OA14OA15 OA16 C. elegans, GnTI, Δ35 PA12 PA13 PA14 PA15 PA16 C. elegans,GnTI, Δ63 PB12 PB13 PB14 PB15 PB16 X. leavis, GnTI, Δ33 QA12 QA13 QA14QA15 QA16 X. leavis, GnTI, Δ103 QB12 QB13 QB14 QB15 QB 16

Targeting peptide sequences were selected from OCHI in P. pastoris(long, medium and short) (see Example 11) and MNN9 (SwissProt P39107) inS. cerevisiae short, and medium. Catalytic domains were selected fromhuman GnTI with a 38 and 86 amino acid N-terminal deletion, C. elegans(gly-12) GnTI with a 35 and 63 amino acid deletion as well as C. elegans(gly-14) GnTI with a 88 amino acid N-terminal deletion and X. leavisGnTI with a 33 and 103 amino acid N-terminal deletion, respectively.

A portion of the gene encoding human N-acetylglucosaminyl Transferase I(MGATI, Accession# NM002406), lacking the first 154 bp, was amplified byPCR using oligonucleotides 5′-TGGCAGGCGCGCCTCAGTCAGCGCTCTCG-3′ (SEQ IDNO:32) and 5′-AGGTTAATTA AGTGCTAATTCCAGCTAGG-3′ (SEQ ID NO:33) andvector pHG4.5 (ATCC#79003) as template. The resulting PCR product wascloned into pCR2.1-TOPO and the correct sequence was confirmed.Following digestion with AscI and PacI the truncated GnTI was insertedinto plasmid pJN346 to create pNA. After digestion of pJN271 with NotIand AscI, the 120 bp insert was ligated into pNA to generate an in-framefusion of the MNN9 transmembrane domain with the GnTI, creating pNA 15.

The host organism is a strain of P. pastoris that is deficient inhypermannosylation (e.g. an och1 mutant), provides the substrateUDP-GlcNAc in the Golgi and/or ER (i.e. contains a functional UDP-GlcNActransporter), and provides N-glycans of the structure Man₅GlcNAc₂ in theGolgi and/or ER (e.g. P. pastoris pFB8 (Saccharomyces SEC12 (m)/mousemannosidase IA Δ187) from above). First, P. pastoris pFB8 wastransformed with pPB103 containing the Kluyveromyces lactis MNN2-2 gene(Genbank AN AF106080) (encoding UDP-GlcNAc transporter) cloned intoBamHI and BglII site of pBLADE-SX plasmid (Cereghino et al. Gene 263(2001) 159-169). Then the aforementioned combinatorial DNA libraryencoding a combination of exogenous or endogenous GnTI/localizationgenes was transformed and colonies were selected and analyzed for thepresence of the GnTI construct by colony PCR. Our transformation andintegration efficiency was generally above 80% and PCR screening can beomitted once robust transformation parameters have been established.

Protein Purification

K3 was purified from the medium by Ni-affinity chromatography utilizinga 96-well format on a Beckman BioMek 2000 laboratory robot. The roboticpurification is an adaptation of the protocol provided by Novagen fortheir HisBind resin. Another screening method may be performed using aspecific terminal GlcNAc binding antibody, or a lectin such as the GSIIlectin from Griffonia simplificolia, which binds terminal GlcNAc (EYLaboratories, San Mateo, Calif.). These screens can be automated byusing lectins or antibodies that have been modified with fluorescentlabels such as FITC or analyzed by MALDI-TOF.

Secreted K3 can be purified by Ni-affinity chromatography, quantifiedand equal amounts of protein can be bound to a high protein binding96-well plate. After blocking with BSA, plates can be probed with aGSII-FACS lectin and screened for maximum fluorescent response. Apreferred method of detecting the above glycosylated proteins involvesthe screening by MALDI-TOF mass spectrometry following the affinitypurification of secreted K3 from the supernatant of 96-well culturedtransformants. Transformed colonies were picked and grown to an OD600 of10 in a 2 ml, 96-well plate in BMGY at 30° C. Cells were harvested bycentrifugation, washed in BMMY and resuspended in 250 ul of BMMY.Following 24 hours of induction, cells were removed by centrifugation,the supernatant was recovered and K3 was purified from the supernatantby Ni affinity chromatography. The N-glycans were released and analyzedby MALDI-TOF delayed extraction mass spectrometry as described herein.

In summary, the methods of the invention yield strains of P. pastoristhat produce GlcNAcMan₅GlcNAc₂ in high yield, as shown in FIG. 10B. Atleast 60% of the N-glycans are GlcNAcMan₅GlcNAc₂. To date, no reportexists that describes the formation of GlcNAcMan₅GlcNAc₂ on secretedsoluble glycoproteins in any yeast. Results presented herein show thataddition of the UDP-GlcNAc transporter along with GnTI activity producesa predominant GlcNAcMan₅GlcNAc₂ structure, which is confirmed by thepeak at 1457 (m/z) (FIG. 10B).

Construction of Strain PBP-3:

The P. pastoris strain expressing K3, (Δoch1, arg-, ade-, his-) wastransformed successively with the following vectors. First, pFB8(Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) was transformed inthe P. pastoris strain by electroporation. Second, pPB103 containingKluyveromyces lactis MNN2-2 gene (Genbank AN AF106080) (encodingUDP-GlcNAc transporter) cloned into pBLADE-SX plasmid (Cereghino et al.Gene 263 (2001) 159-169) digested with BamHI and BglII enzymes wastransformed in the P. pastoris strain. Third, pPB104 containingSaccharomyces MNN9(s)/human GnTI Δ38 encoding gene cloned as NotI-PacIfragment into pJN336 was transformed into the P. pastoris strain.

EXAMPLE 16 Engineering K. lactis Cells to Produce N-Glycans with theStructure Man₅GlcNAc₂

Identification and Disruption of the K. lactis OCH1 Gene

The OCH1 gene of the budding yeast S. cerevisiae encodes a1,6-mannosyltransferase that is responsible for the first Golgilocalized mannose addition to the Man₈GlcNAc₂ N-glycan structure onsecreted proteins (Nakayama et al, J Biol. Chem.; 268(35):26338-45 (Dec.15, 1993)). This mannose transfer is generally recognized as the keyinitial step in the fungal specific polymannosylation of N-glycanstructures (Nakanishi-Shindo et al, 1993; Nakayama et al, 1992;Morin-Ganet et al, Traffic 1(1):56-68. (January 2000)). Deletion of thisgene in S. cerevisiae results in a significantly shorter N-glycanstructure that does not include this typical polymannosylation or agrowth defect at elevated temperatures (Nakayama et al, EMBO J.;11(7):2511-9 (July 1992)).

The Och1p sequence from S. cerevisiae was aligned with known homologsfrom Candida albicans (Genbank accession # AAL49987), and P. pastoris(B. K. Choi et al. in prep) along with the Hoc1 proteins of S.cerevisiae (Neiman et al, Genetics, 145(3):637-45 (March 1997) and K.lactis (PENDANT EST database) which are related but distinctmannosyltransferases. Regions of high homology that were in common amongOch1p homologs but distinct from the Hoc1p homologs were used to designpairs of degenerate primers that were directed against genomic DNA fromthe K. lactis strain MG1/2 (Bianchi et al, Current Genetics 12, 185-192(1987)). PCR amplification with primers RCD33(CCAGAAGAATTCAATTYTGYCARTGG) (SEQ ID NO:34) and RCD34(CAGTGAAAATACCTGGNCCNGTCCA) (SEQ ID NO:35) resulted in a 302 bp productthat was cloned and sequenced and the predicted translation was shown tohave a high degree of homology to Och1 proteins (>55% to S. cerevisiaeOch1p).

The 302 bp PCR product was used to probe a Southern blot of genomic DNAfrom K. lactis strain (MG1/2) with high stringency (Sambrook et al,1989). Hybridization was observed in a pattern consistent with a singlegene indicating that this 302 bp segment corresponds to a portion of theK. lactis genome and K. lactis (KlOCH1) contains a single copy of thegene. To clone the entire KlOCH1 gene, the Southern blot was used to mapthe genomic locus. Accordingly, a 5.2 kb BamHI/PstI fragment was clonedby digesting genomic DNA and ligating those fragments in the range of5.2 kb into pUC19 (New England Biolabs, Beverly, Mass.) to create a K.lactis subgenomic library. This subgenomic library was transformed intoE. coli and several hundred clones were tested by colony PCR using RCD33/34. The 5.2 kb clone containing the predicted KlOCH1 gene wassequenced and an open reading frame of 1362 bp encoding a predictedprotein that is 46.5% identical to the S. cerevisiae OCH1 gene. The 5.2kb sequence was used to make primers for construction of anoch1::KAN^(R) deletion allele using a PCR overlap method (Davidson etal, Microbiology. 148(Pt 8):2607-15. August 2002). This deletion allelewas transformed into two K. lactis strains and G418 resistant coloniesselected. These colonies were screened by both PCR and for temperaturesensitivity to obtain a strain deleted for the OCH1 ORF. The results ofthe experiment show strains which reveal a mutant PCR pattern, whichwere characterized by analysis of growth at various temperatures andN-glycan carbohydrate analysis of secreted and cell wall proteinsfollowing PNGase digestion. The och1 mutation conferred a temperaturesensitivity which allowed strains to grow at 30° C. but not at 35° C.FIG. 12A shows a MALDI-TOF analysis of a wild type K. lactis strainproducing N-glycans of Man₈GlcNAc₂ [a] and higher.

Identification, Cloning, and Disruption of the K. lactis MNN1 Gene

S. cerevisiae MNN1 is the structural gene for the Golgiα-1,3-mannosyltransferase. The product of MNN1 is a 762-amino acid typeII membrane protein (Yip et al., Proc Natl Acad Sci USA. 91(7):2723-7.(1994)). Both N-linked and O-linked oligosaccharides isolated from mnn1mutants lack α-1,3-mannose linkages (Raschke et al., J Biol. Chem.,248(13):4660-6. (Jul. 10, 1973).

The Mnn1p sequence from S. cerevisiae was used to search the K. lactistranslated genomic sequences (PEDANT). One 405 bp DNA sequence encodinga putative protein fragment of significant similarity to Mnn1p wasidentified. An internal segment of this sequence was subsequently PCRamplified with primers KMN1 (TGCCATCTTTTAGGTCCAGGCCCGTTC) (SEQ ID NO:36)and KMN2 (GATCCCACGACGCATCGTATTTCTTTC), (SEQ ID NO:37) and used to probea Southern blot of genomic DNA from K. lactis strain (MG1/2). Based onthe Southern hybridization data a 4.2 Kb BamHI-PstI fragment was clonedby generating a size-selected library as described herein. A singleclone containing the K. lactis MNN1 gene was identified by whole colonyPCR using primers KMN1 (SEQ ID NO:36) and KMN2 (SEQ ID NO:37) andsequenced. Within this clone a 2241 bp ORF was identified encoding apredicted protein that was 34% identical to the S. cerevisiae MNN1 gene.Primers were designed for construction of a mnn1::NAT^(R) deletionallele using the PCR overlap method (Davidson et al. 2002).

This disruption allele was transformed into a strain of K. lactis byelectroporation and Noursethoicin resistant transformants were selectedand PCR amplified for homologous insertion of the disruption allele.Strains that reveal a mutant PCR pattern may be subjected to N-glycancarbohydrate analysis of a known reporter gene.

FIG. 12B depicts the N-glycans from the K. lactis och1 mnn1 deletionstrain observed following PNGase digestion the MALDI-TOF as describedherein. The predominant peak at 1908 (m/z) indicated as [d] isconsistent with the mass of Man₉GlcNAc₂.

EXAMPLE 17 Engineering Plant Cells to Express GlcNAc Transferases orGalactosyltransferases

GlcNAc transferase IV is required for the addition of β1,4 GlcNAc to theα-1,6 mannose residue and the α-1,3 mannose residues in complexN-glycans in humans. So far GlcNAc transferase IV has not been detectedin or isolated from plants. A transgenic plant that is capable of addinghuman-like N-glycans must therefore be engineered to express GlcNActransferase IV. Thus, the plant host cell or transgenic plant must alsolocalize an expressed GlcNAc transferase IV to the correct intracellularcompartment in the host so that the enzyme can add the β1,4 GlcNAc tothe appropriate mannose residues.

There is some evidence that glycosyltransferases from mammals and plantshave similar targeting signals. For example, a full-length ratα2,6-sialyltransferase has been shown to correctly localize to the transGolgi network in transgenic arabidopsis though not necessarily active(Wee E et al. Plant Cell 1998 October; 10(10): 1759-68). A fusionconstruct having fifty-two N-terminal amino acids fromα2,6-sialyltransferase fused to a green fluorescent reporter protein(GFP) was also shown to correctly localize to the plant Golgi (Boevinket al. Plant J 1998 August; 15(3):441-7). Two mammalian proteins—TGN30and furin—and AtELP, an arabidopsis integral membrane protein(Sanderfoot et al. Proc Natl Acad Sci USA 1998 Aug. 18; 95(17):9920-5),which localize to the trans Golgi network, each contain a tyrosinetetrapeptide motif which targets them to the Golgi, probably by arecycling mechanism via the plasma membrane. Although mammals and plantsappear to share some common mechanisms related to protein targeting,exogenous glycosylases may nonetheless not target correctly in a plantcell, however, localization does not necessarily equal enzyme activity.It therefore becomes essential to devise means to correctly target in aplant cell these enzymes and/or other enzymes which participate informing complex, human-like N-glycans.

Glycosylation enzymes are integral membrane proteins which reside in theendoplasmic reticulum and Golgi apparatus. The targeting andlocalization signals are normally contained in the cytoplasmic and/ortransmembrane domains and in some cases are contained in some lumenalamino acids. For example, fifty-two amino acids that make up thetransmembrane domain, nine cytoplasmic amino acids and twenty-sixlumenal amino acids of α2,6-sialyltransferase are required to target GFPto the trans Golgi network (Boevink et al. Plant J 1998 August;15(3):441-7).

Thus, a library of sequences encoding cellular targeting signal peptidescomprising of either just the cytoplasmic and transmembrane domains orthe cytoplasmic, transmembrane and lumenal domains of endoplasmicreticulum and Golgi specific proteins is generated, as described inExample 11. The targeting peptide sequences maybe chosen from ER andGolgi-resident plant, yeast or animal proteins. A glycosylation relatedprotein, e.g., an enzyme (or catalytic domain thereof) such as aglycosylase or integral membrane enzyme can be fused in-frame to thelibrary of targeting peptide sequences and introduced into plants (FIG.13). Plant targeting peptide sequences may be most efficient inlocalizing the chimeric enzymes to the ER and Golgi, although targetingpeptide sequences from fungi and mammals may also be effective. Forexample, the N-terminal 77 amino acids from tobacco N-acetylglucosaminylTransferase I have been shown to correctly target a reporter protein tothe Golgi (Essl D. et al., FEBS Lett 1999 Jun. 18; 453(1-2):169-73). Inone embodiment, one or more N-terminal fragments comprising these 77amino acids (or subsets of these amino acids) is fused to one or morefragments comprising a catalytic domain of GlcNAc transferase IV. Atleast one resulting fusion protein correctly localizes a functionalGlcNAc transferase IV to the Golgi apparatus in a plant cell, asevidenced by monitoring the glycosylation state of a reporterglycoprotein resident or introduced into the plant host cell usingtechniques described herein.

Another plant enzyme shown to localize to the Golgi is ArabidopsisGlcNAc transferase II (Strasser R et al., Glycoconj J 1999 December;16(12):787-91). Thus, in another embodiment, one or more differentfragments of the arabidopsis GlcNAc transferase II targeting peptide arefused to a GlcNAc transferase IV catalytic domain and fusion constructsproduced and tested as described above. The plant specificβ1,2-xylosyltransferase from Arabidopsis thaliana is another proteinthat localizes to the Golgi and its localization and retention in theGolgi is dependent on its cytoplasmic and transmembrane sequences(Dirnberger et al., Plant Mol Biol 2002 September; 50(2):273-81). Thus,in another embodiment, one or more fragments comprising the cytoplasmicand transmembrane sequences of β1,2-xylosyltransferase are fused to oneor more fragments comprising a GlcNAc transferase IV catalytic domainand resulting fusion constructs are transformed into plant cells andtested for their ability to produce a human-like N-glycan and tootherwise modulate glycosylation in the plant host cell.

Because GlcNAc transferase IV or Galactosyltransferase from one organismmay function more efficiently in a specific plant host than one fromanother organism, fragments comprising GlcNAc transferase IVs (orcatalytic domains) from various eukaryotic organisms are fused in-frameto the library of endoplasmic reticulum (ER) and Golgi targeting peptidesequences and are then introduced into plants. The use of a library ofnucleic acids encoding enzyme domains isolated or derived from differentspecies increases the chances of efficient glycosylation—in addition tocorrect localization and glycosylation by GlcNAc transferase IV.

The methods and combinatorial nucleic acid libraries of the inventionmay be used to introduce and localize, sequentially or en masse,multiple enzymes required to glycosylate proteins in a plant cell withhuman-like N-glycans. As different plant species may require differentgrowth conditions, protocols for transformation may vary depending onthe species being transformed (Potrykus, “Gene transfer methods forplants and cell cultures.” Ciba Found Symp 1990; 154:198-208; discussion208-12). The commonly used methods for generating transgenic plantsinclude Agrobacterium mediated transformation, particle bombardment(Sanford, J. C. et al, Biolistic plant transformation. Physiol. Plant.1990, 79: 206-209) and electroporation.

Agrobacterium Method

The catalytic domains of GlcNAc transferase IVs are fused in-frame tomultiple different targeting peptide sequences known to target proteinsto the ER and Golgi in plants. Each of these fusion constructs isintroduced under the control of the ubiquitously expressed promoterslike the 35S CaMV, ubiquitin or actin promoters, tissue specificpromoters or inducible promoters. A plant specific terminator region isalso used. This cassette (Promoter::targeting peptide-GlcNAc transferaseIV::terminator) is cloned into a vector suitable for Agrobacteriummediated transformation (FIG. 13). The vector also contains a selectablemarker that allows one to select for transformed plants. The commonselectable markers used include those resulting in kanamycin, hygromycinand basta resistance. The construct is introduced into Agrobacterium viawell-established transformation methods, which are available in the art.An Agrobacterium library of Golgi-targeted GlcNAc transferase IVs isthereby generated.

Embryonic and meristematic tissue may be transformed and can regeneratetransgenic plants. To transform tissue, tissue explants (these could beplumules and radicals from germinated seeds) are first soaked and coatedwith an Agrobacterium innoculum. They are then cultured on platescontaining the innoculum to form an undifferentiated mass of cellstermed the callus. Transformed plant cells are selected for by adding tothe medium the relevant kanamycin, hygromycin or basta (depending on theselectable marker used on the construct). The transformed plant cellscan either be grown in culture and remain undifferentiated or they aretreated with shoot regenerating and shoot elongation medium. Explantsthat differentiate are transferred onto rooting medium to generatetransgenic plants. Some plants like Arabidopsis can be transformed bydipping flowers into an Agrobacterium solution. Seeds from thetransformed plants are germinated on plates containing the relevantherbicide or antibiotic selection. Transgenic plants are those that growon the selection media. The transgenic plants are then screened forthose with properly glycosylated proteins (i.e., those which havecomplex, human-like N-glycans) by isolating glycoproteins from plantextracts and analyzing glycoprotein patterns as described elsewhereherein, e.g., by using a specific antibody or lectin. Although theAgrobacterium method is economical and simple, it is limited to certainspecies of plants. Accordingly, plants that cannot be transformed usingAgrobacterium can be transformed by ballistics or electroporation.

Particle Bombardment Method and Electroporation

Compared to Agrobacterium mediated transformation, these methods have agreater tendency to insert multiple copies of the transgene into thegenome. This could result in gene silencing and cosuppression. However,unlike Agrobacterium mediated transformation, these methods are notspecies limited and are therefore useful when an Agrobacterium methodcannot be employed to generate transgenic plants. In the particlebombardment method, cultured plant cells are bombarded with very smalltungsten or gold particle that have been coated with DNA(Promoter::targeting peptide-GlcNAc transferaseIV-terminator::selectable marker) (FIG. 13) (rb and lb not required)while in the electroporation method, plant cells in a DNA(Promoter::targeting peptide-GlcNAc transferaseIV-terminator::selectable marker) solution are treated with an electricpulse that perforates the cell, allowing it to take up DNA. The cellsare then cultured and allowed to recover. Stable transformants areselected for by culturing and regenerating plants on appropriateselection medium.

Engineering Soybeans to Express GlcNAc Transferase IV Using a SoybeanCotyledonary Node Agrobacterium Mediated Transformation System

An Agrobacterium library of Golgi-targeted GlcNAc transferase IV isgenerated as described above. Soybean explants are transformed with thelibrary using a protocol described by Hinchee et al (Bio/Technology1988. 6:915). A reporter protein is expressed with a His tag, purifiedand then analyzed. Transgenic plants are assayed for proteins with theα-1,6 mannose and the α-1,3 mannose residues using, e.g., massspectroscopy.

Engineering Pea to Express GlcNAc Transferase IV Using ParticleBombardment

A GlcNAc transferase IV plasmid library is coated onto tungsten or goldparticles and used as microprojectiles to bombard calli derived from peaembryonic tissue as described (Molnar et al., Symposium on RecentAdvances in Plant Biotechnology, Sep. 4-11, 1999, Stara Lesna, SlovakRepublic). A reporter protein is expressed with a His tag, purified andthen analyzed. Transgenic plants are assayed for proteins with the α-1,6mannose and the α-1,3 mannose residues using, e.g., MALDI.

Engineering Plants to Express GlcNAc Transferase I

GlcNAc transferase I is involved in the addition of GlcNAc to theterminal α-1,3 mannose residue to form Man₅GlcNAc₂, an essential step inthe maturation of complex N-glycans. Although GlcNAc transferase I hasbeen isolated from plants and appears to have the same function as itsmammalian homolog, it may not be the most efficient enzyme forglycosylation of mammalian or exogenous proteins and may not be found inevery plant species. As the addition of GlcNAc to the terminal α-1,3mannose residue is a controlling step in the mammalian glycosylationpathway, it is advantageous to have transgenic plants that can carry outthis step efficiently. To create transgenic plants that express GlcNActransferase I that can function efficiently to promote the formation ofcomplex N-glycans, a library of GlcNAc transferase I isolated or derivedfrom various organisms is fused in-frame to multiple plant Golgitargeting peptide sequences according to the methods described herein.The combinatorial library thus created is introduced into a plant cellor organism as described above for GlcNAc transferase IV.

Engineering Maize to Express GlcNAc Transferase I Using ParticleBombardment

Transgenic maize can be obtained using a protocol similar to the oneused to generate peas that express GlcNAc transferase IV. Here theGlcNAc transferase I plasmid library is coated onto tungsten or goldparticles and used to bombard calli derived from maize embryonic tissue,e.g., using a protocol specific for the generation of transgenic maize(Gordon-Kamm W J et al., Plant Cell 1990 July; 2(7):603-618)).Transgenic plants are assayed for proteins having GlcNAc on the terminalα-1,3 mannose residue, e.g., using specific antibodies or by assayingreduced binding of the N-glycans to certain lectins or by usingMALDI-TOF.

Other useful references for using plant host cells according to theinvention include: Christou P. Plant Mol Biol 1997 September;35(1-2):197-203; Chowrira G M et al. Mol Biotechnol 1995 February;3(1):17-23; Dirnberger et al., Plant Mol Biol 2002 September;50(2):273-81; Frame B R et al. Plant Physiol 2002 May; 129(1):13-22;Gomord V et al. Biochimie 1999 June; 81(6):607-18; Laursen C M et al.Plant Mol Biol 1994 January; 24(1):51-61; Orci L et al. J Cell Biol 2000Sep. 18; 150(6):1263-70; Newell Calif. Mol Biotechnol 2000 September;16(1):53-65; Pawlowski Wpet al. Mol Biotechnol 1996 August; 6(1):17-30;Schroeder H E et al. Plant Physiol 1993 March; 101(3):751-757; Sorokin,A P et al. Plant Sci. 2000 Jul. 28; 156(2):227-233; Strasser R et al.Glycoconj J 1999 December; 16(12):787-91; and Tomes D T et al. Plant MolBiol 1990 February; 14(2):261-8.

Engineering Plant Cells to Produce β1,4-Galactosyltransferases

β1,4-galactosyltransferase is an important human glycosyltransferasethat is absent in plants. Lerouge P et al. Plant Mol Biol 1998September; 38(1-2):31-48. In mammals, β1,4-galactosyltransferase islocalized in the Golgi and is responsible for the transfer of galactoseresidues to the terminal N-acetylglucosamine of the core Man₃GlcNAc₂ ofcomplex N-glycans. In plants, the Man₃GlcNAc₂ core contains β1,2-xyloseand α1,3-fucose residues and lacks the β1,4-galactose. The xylose andfucose modifications are implicated in allergies and act as antigenicepitopes and are therefore not desirable modifications of therapeuticproteins.

The galactose modifications carried out by β1,4-galactosyltransferasecan be important for the proper functioning of the therapeutic proteins.In mammals, β1,4-galactosyltransferase acts afterN-acetylglucosaminyltransferase I and N-acetylglucosaminyltransferase IIand has been shown to initiate branching of the complex N-glycan.Lerouge P et al. Plant Mol Biol 1998 September; 38(1-2):31-48. PalacpacN et al. Proc Natl Acad Sci USA 1999 Apr. 13; 96(8):4692-7. In tobaccocells, expression of human β1,4-galactosyltransferase has been shown toresult in galactosylated N-glycans with reduced fucose and xylosemodifications. Bakker H et al. Proc Natl Acad Sci USA 2001 Feb. 27;98(5):2899-904 Fujiyama K et al. Biochem Biophys Res Commun 2001 Nov.30; 289(2):553-7. Palacpac N et al. Proc Natl Acad Sci USA 1999 Apr. 13;96(8):4692-7. In these studies, a 1.2 kb fragment of humanβ1,4-galactosyltransferase was cloned downstream of the cauliflowermosaic virus promoter (35SCaMV), introduced into the binary vectorpGA482, and finally into tobacco cells. Palacpac N et al. Proc Natl AcadSci USA 1999 Apr. 13; 96(8):4692-7.

Tobacco cells were transformed using the agrobacterium method describedby Rempel et al. (Rempel, H. C. et al. 1995. Transgenic Res.4(3):199-207.) Transformation of tobacco cells has also been described(An, G 1985. Plant Physiol. 79:568-570). Expression ofβ1,4-galactosyltransferase under the 35SCaMV resulted in ubiquitousexpression of the gene in tobacco cells. Tobacco cells expressing humanβ1,4-galactosyltransferase showed the presence of galactosylatedN-glycans. (Palacpac N et al. Proc Natl Acad Sci USA 1999 Apr. 13;96(8):4692-7). Bakker et al. showed that crossing tobacco plantsexpressing human β1,4-galactosyltransferase with plants expressing theheavy and light chain of a mouse antibody resulted in plants in whichthe antibody showed 30% galactosylation (Bakker H et al. Proc Natl AcadSci USA 2001 Feb. 27; 98(5):2899-904).

A combinatorial DNA library can be constructed to obtain aβ1,4-galactosyltransferase line for the addition of galactose residues.The combinatorial DNA library can effectively produce lines which aremore efficient in the addition of galactose residues. Once such a lineis made it can be easily crossed to lines expressing other glycosylationenzymes and to those expressing therapeutic proteins to producetherapeutic proteins with human-like glycosylation. The final line canthen be grown as plants and harvested to extract proteins or can becultured as plant cells in suspension cultures to produce proteins inbioreactors. By expressing the therapeutic proteins using the library ofsignal peptides, it is possible to retain the therapeutic protein withinthe cells or have them secreted into the medium. Tobacco cellsexpressing β1,4-galactosyltransferase secrete galactosylated N-glycans(Ryo Misaki et al. Glycobiology 2002 Dec. 17; 10:1093). Whilehorseradish peroxidase isozyme C expressed in tobacco plants expressingβ1,4-galactosyltransferase contained xylose and fucose modifications, noxylose or fucose modification could be detected in horseradishperoxidase isozyme C expressed in tobacco cells expressingβ1,4-galactosyltransferase (GT6 cells). (Fujiyama K et al. BiochemBiophys Res Commun 2001 Nov. 30; 289(2):553-7). This indicates that itmay be advantageous to express therapeutic proteins in cell linesinstead of whole plants.

Engineering Plants to Produce Sialyltransferase

In mammals, sialyltransferase is a trans golgi enzyme that adds terminalsialic acid residues to glycosylated polypeptides. Thus far, terminalsialic acid residues have not been detected in plants (Wee E et al.Plant Cell 1998 October; 10(10):1759-68). Wee et al. expressed the ratα2,6-sialyltransferase in transgenic arabidopsis and showed that theenzyme properly localized to the golgi and was functional. Wee et al.demonstrated that membranes isolated from transgenic arabidopsis, whenincubated with CMP-³H-sialic acid and asialofetuin acceptor, resulted inthe addition of sialic acid residues while membrane isolated fromwild-type arabidopsis did not. While expressing the ratα2,6-sialyltransferase in arabidopsis resulted in a functional enzymethat was able to incorporate sialic acid residues, fusing the mammalianenzymes α-2,3-sialyltransferase and α2,6-sialyltransferase to a varietyof transit peptides using the library approach of the present inventiondescribed earlier can result in more efficient sialylation in otherplant species. Wee E et al had to isolate membranes and incubate themwith CMP-³H-sialic acid and asialofetuin acceptor since arabidopsis doesnot have CMP-sialic acid or its transporter. In order to overcome thisadditional step and obtain sialic acid addition in the plant, CMP-sialicacid biosynthetic pathway and the CMP-sialic acid transporter can beco-expressed in transgenic plants expressing α-2,3-sialyltransferase andα2,6-sialyltransferase. As an alternative the CMP-sialic acidtransporter can be co-expressed α-2,3-sialyltransferase andα2,6-sialyltransferase in plant cells grown in suspension culture, andCMP-sialic acid or other precursors of CMP-sialic acid supplied in themedium.

Expressing α-2,3-Sialyltransferase and α-2,6-Sialyltransferase in Lemna

As described in the U.S. Pat. No. 6,040,498, lemna (duckweed) can betransformed using both agrobacterium and ballistic methods. Usingprotocols described in the patent, lemna will be transformed with alibrary of golgi targeted α-2,3-sialyltransferase and/orα-2,6-sialyltransferase and a library of mammalian CMP-sialic acidtransporters. Transgenic plants can be assayed for proteins withterminal sialic acid residues.

Expressing α-2,3-Sialyltransferase and α-2,6-Sialyltransferase inTobacco Cells

Alpha-2,3-sialyltransferase and/or α-2,6-sialyltransferase and/or alibrary of mammalian CMP-sialic acid transporters can also be introducedinto tobacco cells grown in suspension culture as described forβ1,4-galactosyltransferases. CMP-sialic acid can be added to the medium.Both the cells and the culture medium (secreted proteins) can be assayedfor proteins with terminal sialic acid residues.

EXAMPLE 18 Engineering Insect Cells to Produce Glycosyltransferases

Insect cells provide another mechanism for producing glycoproteins butthe resulting glycoproteins are not complex human-like glycoproteins.Marz et al. 1995 Glycoproteins, 29:543-563; Jarvis 1997 TheBaculoviruses 389-431. It is another feature of the present invention toprovide enzymes in insect cells, which are targeted to the organelles inthe secretory pathway. In a preferred embodiment, enzymes such asglycosyltransferases, galactosyltransferases and sialyltransferases aretargeted to the ER, Golgi or the trans Golgi network in lepidopteraninsect cells (Sf9). Expression of mammalian β1,4-galactosyltransferasehas been shown in Sf9 cells. Hollister et al. Glycobiology. 19988(5):473-480. These enzymes are targeted by means of a chimeric proteincomprising a cellular targeting signal peptide not normally associatedwith the enzyme. The chimeric proteins are made by constructing anucleic acid library comprising targeting sequences as described hereinand the glycosylation enzymes. Baculovirus expression in insect cells iscommonly used for stable transformation for adding mammalianglycosyltransferases in insect cells. Hollister et al. Glycobiology.2001 11(1):1-9.

TABLE 11 DNA and Protein Sequence Resources  1. European BioinformaticsInstitute (EBI) is a centre for research and services in bioinformatics:http://www.ebi.ac.uk/  2. Swissprot database: http://www.expasy.ch/spr 3. List of known glycosyltransferases and their origin.  4. human cDNA,Kumar et al (1990) Proc. Natl. Acad. Sci. USA 87: 9948-9952  5. humangene, Hull et al (1991) Biochem. Biophys. Res. Commun. 176: 608-615  6.mouse cDNA, Kumar et al (1992) Glycobiology 2: 383-393  7. mouse gene,Pownall et al (1992) Genomics 12: 699-704  8. murine gene (5′ flanking,non-coding), Yang et al (1994) Glycobiology 5: 703-712  9. rabbit cDNA,Sarkar et al (1991) Proc. Natl. Acad. Sci. USA 88: 234-238 10. rat cDNA,Fukada et al (1994) Biosci. Biotechnol. Biochem. 58: 200-201 1,2 (GnTII)EC 2.4.1.143 11. human gene, Tan et al (1995) Eur. J. Biochem. 231:317-328 12. rat cDNA, D'Agostaro et al (1995) J. Biol. Chem. 270:15211-15221 13. β1, 4 (GnTIII) EC 2.4.1.144 14. human cDNA, Ihara et al(1993) J. Biochem. 113: 692-698 15. murine gene, Bhaumik et al (1995)Gene 164: 295-300 16. rat cDNA, Nishikawa et al (1992) J. Biol. Chem.267: 18199-18204 β1, 4 (GnTIV) EC 2.4.1.145 17. human cDNA, Yoshida etal (1998) Glycoconjugate Journal 15: 1115-1123 18. bovine cDNA, Minowaet al. , European Patent EP 0 905 232 β1, 6 (GnT V) EC 2.4.1.155 19.human cDNA, Saito et al (1994) Biochem. Biophys. Res. Commun. 198:318-327 20. rat cDNA, Shoreibah et al (1993) J. Biol. Chem. 268:15381-15385 131, 4 Galactosyltransferase, EC 2.4.1.90 (LacNAcsynthetase) EC 2.4.1.22 (lactose synthetase) 21. bovine cDNA, D'Agostaroet al (1989) Eur. J. Biochem. 183: 211-217 22. bovine cDNA (partial),Narimatsu et al (1986) Proc. Natl. Acad. Sci. USA 83: 4720-4724 23.bovine cDNA (partial), Masibay & Qasba (1989) Proc. Natl. Acad. Sci. USA86: 5733-5377 24. bovine cDNA (5′ end), Russo et al (1990) J. Biol.Chem. 265: 3324 25. chicken cDNA (partial), Ghosh et al (1992) Biochem.Biophys. Res. Commun. 1215-1222 26. human cDNA, Masri et al (1988)Biochem. Biophys. Res. Commun. 157: 657-663 27. human cDNA, (HeLa cells)Watzele & Berger (1990) Nucl. Acids Res. 18: 7174 28. human cDNA,(partial) Uejima et al (1992) Cancer Res. 52: 6158-6163 29. human cDNA,(carcinoma) Appert et al (1986) Biochem. Biophys. Res. Commun. 139:163-168 30. human gene, Mengle-Gaw et al (1991) Biochem. Biophys. Res.Commun. 176: 1269-1276 31. murine cDNA, Nakazawa et al (1988) J.Biochem. 104: 165-168 32. murine cDNA, Shaper et al (1988) J. Biol.Chem. 263: 10420-10428 33. murine cDNA (novel), Uehara & Muramatsuunpublished 34. murine gene, Hollis et al (1989) Biochem. Biophys. Res.Commun. 162: 1069-1075 35. rat protein (partial), Bendiak et al (1993)Eur. J. Biochem. 216: 405-417 2,3-Sialyltransferase, (ST3Gal II)(N-linked) (Gal-1,3/4-GlcNAc) EC 2.4.99.6 36. human cDNA, Kitagawa &Paulson (1993) Biochem. Biophys. Res. Commun. 194: 375-382 37. rat cDNA,Wen et al (1992) J. Biol. Chem. 267: 21011-21019 2,6-Sialyltransferase,(ST6Gal I) EC 2.4.99.1 38. chicken, Kurosawa et al (1994) Eur. J.Biochem 219: 375-381 39. human cDNA (partial), Lance et al (1989)Biochem. Biophys. Res. Commun. 164: 225-232 40. human cDNA, Grundmann etal (1990) Nucl. Acids Res. 18: 667 41. human cDNA, Zettlmeisl et al(1992) Patent EP0475354-A/3 42. human cDNA, Stamenkovic et al (1990) J.Exp. Med. 172: 641-643 (CD75) 43. human cDNA, Bast et al (1992) J. CellBiol. 116: 423-435 44. human gene (partial), Wang et al (1993) J. Biol.Chem. 268: 4355-4361 45. human gene (5′ flank), Aasheim et al (1993)Eur. J. Biochem. 213: 467-475 46. human gene (promoter), Aas-Eng et al(1995) Biochim. Biophys. Acta 1261: 166-169 47. mouse cDNA, Hamamoto etal (1993) Bioorg. Med. Chem. 1: 141-145 48. rat cDNA, Weinstein et al(1987) J. Biol. Chem. 262: 17735-17743 49. rat cDNA (transcriptfragments), Wang et al (1991) Glycobiology 1: 25-31, Wang et al (1990)J. Biol. Chem. 265: 17849-17853 50. rat cDNA (5′ end), O'Hanlon et al(1989) J. Biol. Chem. 264: 17389-17394; Wang et al (1991) Glycobiology1: 25-31 51. rat gene (promoter), Svensson et al (1990) J. Biol. Chem.265: 20863-20688 52. rat mRNA (fragments), Wen et al (1992) J. Biol.Chem. 267: 2512-2518

Additional methods and reagents which can be used in the methods formodifying the glycosylation are described in the literature, such asU.S. Pat. No. 5,955,422, U.S. Pat. No. 4,775,622, U.S. Pat. No.6,017,743, U.S. Pat. No. 4,925,796, U.S. Pat. No. 5,766,910, U.S. Pat.No. 5,834,251, U.S. Pat. No. 5,910,570, U.S. Pat. No. 5,849,904, U.S.Pat. No. 5,955,347, U.S. Pat. No. 5,962,294, U.S. Pat. No. 5,135,854,U.S. Pat. No. 4,935,349, U.S. Pat. No. 5,707,828, and U.S. Pat. No.5,047,335. Appropriate yeast expression systems can be obtained fromsources such as the American Type Culture Collection, Rockville, Md.Vectors are commercially available from a variety of sources.

SEOUENCE LISTINGSSEQ ID NO: 1-6 can be found in U.S. Patent application No. 09/892,591SEQ ID NO: 7Primer: regions of high homology within 1, 6 mannosyltransferases5′-atggcgaaggcagatggcagt-3′ SEQ ID NO: 8Primer: regions of high homology within 1, 6 mannosyltransferases5′-ttagtccttccaacttccttc-3′ SEQ ID NO: 9internal primer: 5′-actgccatctgccttcgccat-3′ SEQ ID NO: 10internal primer: 5′-GTAATACGACTCACTATAGGGC-3′ T7 SEQ ID NO: 11Internal primer: 5′-AATTAACCCTCACTAAAGGG-3′ T3 SEQ ID NO: 12Primer: atgcccgtgg ggggcctgtt gccgctcttc agtagc SEQ ID NO: 13Primer: tcatttctct ttgccatcaa tttccttctt ctgttcacgg SEQ ID NO: 14Primer: ggcgcgccga ctcctccaag ctgctcagcg gggtcctgtt ccac SEQ ID NO: 15Primer: ccttaattaa tcatttctct ttgccatcaa tttccttctt ctgttcacggSEQ ID NO: 16Primer: ggcgagctcg gcctacccgg ccaaggctga gatcatttgt ccagcttcagaSEQ ID NO: 17Primer: gcccacgtcg acggatccgt ttaaacatcg attggagagg ctgacaccgc tactaSEQ ID NO: 18Primer: cgggatccac tagtatttaa atcatatgtg cgagtgtaca actcttccca catggSEQ ID NO: 19Primer: ggacgcgtcg acggcctacc cggccgtacg aggaatttct cggatgactc ttttcSEQ ID NO: 20 Primer: cgggatccct cgagagatct tttttgtaga aatgtcttgg tgcctSEQ ID NO: 21Primer: ggacatgcat gcactagtgc ggccgccacg tgatagttgt tcaattgatt gaaataggga caaSEQ ID NO: 22Primer: ccttgctagc ttaattaacc gcggcacgtc cgacggcggc ccacgggtcc caSEQ ID NO: 23Primer: ggacatgcat gcggatccct taagagccgg cagatgcaa attaaagcct tcgagcgtcc cSEQ ID NO: 24Primer: gaaccacgtc gacggccatt gcggccaaaa ccttttttcc tattcaaaca caaggcattg cSEQ ID NO: 25 Primer: ctccaatact agtcgaagat tatcttctac ggtgcctgga ctcSEQ ID NO: 26Primer: tggaaggttt aaacaaagct agagtaaaa tagatatagc gagattagag aatgSEQ ID NO: 27Primer: aagaattcgg ctggaaggcc ttgtaccttg atgtagttcc cgttttcatcSEQ ID NO: 28Primer: gcccaagccg gccttaaggg atctcctgat gactgactca ctgataataa aaatacggSEQ ID NO: 29Primer: gggcgcgta tttaaatacta gtggatctat cgaatctaaa tgtaagttaa aatctctaaSEQ ID NO: 30 Primer: ggccgcctgc agatttaaat gaattcgg cgcgccttaatSEQ ID NO: 31 Primer: taaggcgcgc cgaattcatt taaatctgca gggcSEQ ID NO: 32 Primer: 5′-tggcaggcgcgcctcagtcagcgctctcg-3′ SEQ ID NO: 33Primer: 5′-aggttaatta agtgctaattccagctagg-3′ SEQ ID NO: 34primer for K. lactis OCH1 gene: ccagaagaat tcaattytgy cartggSEQ ID NO: 35primer for K. lactis OCH1 gene: cagtgaaaat acctggnccn gtccaSEQ ID NO: 36primer for K. lactis MNN1 gene: tgccatcttt taggtccagg cccgttcSEQ ID NO: 37primer for K. lactis MNN1 gene: gatcccacga cgcatcgtat ttctttcSEQ ID NO: 38DNA sequence of the 302 bp segment of the putative KlOCH1 gene: gcccttcagtgaaaatacctggcccggtccagttcataatatcggtaccatctgtatttttggcggttttcttttgttgatgtttgtaatttttgttgaacttctttttatccctcatgttgacattataatcatctgcaatgtatttaatacttcagcatcatctaaaggaatgctgcttttaacatttgccacgctctccaatgttgttgcggtgatatttgtgatcaattcgcgcaataatggatggccagattttgattgtattgtccactgacaaaattgaattctaggaagggc SEQ ID NO: 39Translation of putative KlOCH1 gene (excluding primers): TIQSKSGHPLLRELITNITATTLESVANVKSSIPLDDAEVLKDIADDYNVNMRDKKKFNKNYKHQQKKTAKNTDGTDIMN SEQ ID NO: 40DNA sequence of the 405 bp segment of the putative KlMNN1 gene: cccagcgtgccattaccgtatttgccgccgtttgaaatactcaatattcatgatggttgtaaggcgttattatcattcgcgatataatatgccatcattaggtccaggcccgttctcttagctatctaggtgtctgtgctaccgtgatatggtacctattctttttccagtctaatctgaagatggcagatttgaaaaaggtagcaacttcaaggtatctttcacaagaaccgtcgttatcagaacttatgtcaaatgtgaagatcaagcctattgaagaaaccccggtttcgccattggagttgattccagatatcgaaatatcgactagaaagaaatacgatgcgtcgtgggatctgttgttccgtggtagaaaatataaatcgttcaacgattatgatSEQ ID NO: 41 DNA sequence of the K. lactis OCH1 gene: atggggttaccaaagatttcaagaagaacgaggtacattattgtcattgtgctgatactgtacttattgttttctgtgcaatggaatactgcgaaagtgaatcaccatttctataacagcattggcacggtgcttcccagtacagctcgcgtggatcacttgaacttgaaaaacttggacttagcaggtacgagcaataacggtgatcatttgatggatctacgagttcaattggctagtcaattcccctacgattacgagtacccatccccaaaaaggtatggcagacctggaagattgatcccagttcaaagtcacaggtttatccatttcaaaatgccagaatgattggaaacatttcagtgcatccgaggaaccgccatatcaataccaattaatcacagatgatcaaatgataccacttctagagcagctatatggtggggtcccacaagtgataaaggatttgaatccttgccacttccaattcttaaagcagactttttcagatacttgatcctttatgcaagaggtggtatatattctgacatggatacgttcccattaaagccattgtcgtcatggccatcgacttctcagtcctacttttctagtttaaagaatccacaaaggtatagaaattccttggacaaccttgaaacgctagaagatcagaacctggattgtcattggtatcgaggctgatccggatagaagcgattgggcagagtggtacgccaggagaatacaattctgtcagtggacaatacaatcaaaatctggccatccattattgcgcgaattgatcacaaatatcaccgcaacaacattggagagcgtggcaaatgttaaaagcagcattcctttagatgatgctgaagtattaaaagacattgcagatgattataatgtcaacatgagggataaaaagaagttcaacaaaaattacaaacatcaacaaaagaaaaccgccaaaaatacagatggtaccgatattatgaactggactggtccaggtattttttcagatgttattttccagtatcttaataacgttatccagaagaatgatgacattttaatatcaatgataatataatgttatcaacaaacatggatccaaacatgatacaactatgagattctataaagacattgttaaaaatttacaaaacgacaaaccctcattgttctggggattcttttcattgatgacagagcctattctagtggacgacatcatggtacttccgattacttattctcaccaggtatcagaacaatgggcgctaaagaagacaacgacgagatggcatttgttaagcatatttttgaaggaagttggaaagactgaSEQ ID NO: 42 Translation of putative K. lactis OCH1 gene: MGLPKISRRTRYIIVIVLILYLLFSVQWNTAKVNHHFYNSIGTVLPSTARVDHLNLKNLDLAGTSNNGDHLMDLRVQLASQFPYDSRVPIPKKVWQTWKlDPSSKSQVSSISKCQNDWKHFSASEEPPYQYQLITDDQMIPLLEQLYGGVPQVIKAFESLPLPILKADFFRYLILYARGGIYSDMDTFPLKPLSSWPSTSQSYFSSLKNPQRYRNSLDNLETLEASEPGFVIGIEADPDRSDWAEWYARRIQFCQWTIQSKSGHPLLRELITNITATTLESVANVKSSIPLDDAEVLKDIADDYNVNMRDKKKFNKNYKHQQKKTAKNTDGTDIMNWTGPGIFSDVIFQYLNNVIQKNDDILIENDNLNVINKHGSKHDTTMREYKDIVKNLQNDKPSLFWGFFSLMTEPILVDDIMVLPITSFSPGIRTMGAKEDNDEMAFVKHIFEGSWKDZ SEQ ID NO: 43DNA sequence of the K. lactis MNN1 gene: atgatggttgtaaggcgttttttatcagcttcgcgatataatatgccatcttttaggtccaggcccgttctcttagctatctttggtgtctgtgctaccgtgatatggtacctattattnccagtctaatctgaagatggcagatttgaaaaaggtagcaacttcaaggtatctttcacaagaaccgtcgttatcagaacttatgtcaaatgtgaagatcaagcctattgaagaaaccccggtttcgccattggagttgattccagatatcgaaatatcgactagaaagaaatacgatgcgtcgtgggatctgttgttccgtggtagaaaatataaatcgttcaacgattatgatcttcatacgaaatgtgagttttatttccagaatttatacaatttgaacgaggattggaccaataatattcggacgttcactttcgatattaacgatgtagacacgtctacgaaaattgacgctcttaaagattccgatggggttcaattggtggacgagaaggctatacgtttatacaagagaacgcataacgttgccttggctacggaaaggttacgtctttatgataaatgttttgtcaatagtccaggttcaaacccattgaaaatggatcaccttttcagatcgaacaagaagagtaagactacggctttggatgacgaagtcactgggaaccgtgatacttttaccaagacgaagaaaacttcgttcttaagcgatatggacacgagtagtttccagaagtacgatcaatgggatttcgaacatagaatguccccatgatcccatatttgaggaacacaatttcaccaacgtgatgcctattttcaccggctcaaacggtggggaacctttacctcaagggaaattcccggtattagatccaaaatccggtgaattgttacgtgtagagactttcagatatgataaatcgaaatcgctttggaagaactggaatgatatgtcctctgcttctggtaaacgtggtattatcttggctgctggcgacggccaagtggaccaatgcatccgtatattgctacgttgagagctcaaggaaacgctctacctattcaaattatccacaacaaccaattgaatgagaaatctgtgaaactgttatcggaggccgctaaatctaccgaattctcatccggtagagctcaatctattggttagtgaatgtgggccccacgttggaatcttcaatgaagagcaattttgggagatttaagaataagtggttgtcagttattttcaacacttttgaagaatttatattcatagatacagatgccatctcctacattaatatggctgattatttcaacttcaaggagtacaaatctactggaacactcttctttaaggataggtattggcaattggaactgaacagaaatgtggtcattgttcgaaactatgaaccaagaattcttgaaatgtactatttcaatactttacctatgatcaatggtgattacgtggaacagcaatgtatgggcatgctcaccccagaggaaaaagtttacaaacgtttctttgaagttggtcatcaacacaacttggaaagtggattattggccatcaacaaaaacgaacacatcatgggattggttactgcaacagtcttaaatatcgcaccaaaggtcggaggttgcggaggggtgacaaagagtttttctggcttggtttgttggttgctggccaacgctactcgatctatgatatagatgcaagtgcaattggtgttcctcaacagaagcaatctatcgctaacggagacgaatttgatgaatataggatttgttctttacaagtggcacatacttcatacgacggacatttactatggataaatggtggctctcagtactgtaagaaaccagagacttttgaaggtgattggaccaacattaaggagatcgtgaatcgtattctgatgataaagaaaaggctctgaaggcttatagtgatacagttaaggtggaagcagcaatcgtgccagattccagaagtaatggttggggtagagacgatcaaagatgtaaaggctacttctggtgcggcaaatttacttcaaagctgaaaccgtatacttataacacggtggtaactaaaggtgatttgatccgtttcggagacgaggaaatcgaaagtatctccaagattaataagatctggaatgatgctattattccagacggagcttaa SEQ ID NO: 44Translation of putative K. lactis MNN1 gene: MMVVRRFLSASRYNMPSFRSRPVLLAIFGVCATVIWYLFFFQSNLKMADLKKVATSRYLSQEPSLSELMSNVKIKPIEETPVSPLELIPDIEISTRKKYDASWDLLFRGRKYKSFNDYDLHTKCEFYFQNLYNLNEDWTNNIRTFTFDINDVDTSTKIDALKDSDGVQLVDEKAIRLYKRTHNVALATERLRLYDKCFVNSPGSNPLKMDHLFRSNKKSKTTALDDEVTGNRDTFTKTKKTSFLSDMDTSSFQKYDQWDFEHRMFPMIPYFEEHNFTNVMPIFTGSNGGEPLPQGKFPVLDPKSGELLRVETFRYDKSKSLWKNWNDMSSASGKRGIILAAGDGQVDQCIRLIATLRAQGNALPIQIIHNNQLNEKSVKLLSEAAKSTEFSSGRAQSLWLVNVGPTLESSMKSNFGRFKNKWLSVIFNTFEEFIFIDTDAISYINMADYFNFKEYKSTGTLFFKDRSLAIGTEQKCGPLFETLEPRILEMYYENTLPMINGDYVEQQCMGMLTPEEKVYKRFFEVGHQHNLESGLLAINKNEHIMGLVTATVLNIAPKVGGCGWGDKEFFWLGLLVAGQRYSIYDIDASAIGVPQQKQSIANGDEFDEYRKSLQVAHTSYDGHLLWINGGSQYCKKPETFEGDWTNIKELRESYSDDKEKALKAYSDTVKVEAAIVPDSRSNGWGRDDQRCKGYFWCGKFTSKLKPYTYNTVVTKGDLIRFGDEEIESISKlNKlWNDAIIPDGA

REFERENCES

-   Aebi, M., J. Gassenhuber, et al. (1996). “Cloning and    characterization of the ALG3 gene of Saccharomyces cerevisiae.”    Glycobiology 6(4): 439-444.-   Altmann, F., E. Staudacher, et al. (1999). “Insect cells as hosts    for the expression of recombinant glycoproteins.” Glycoconjugate    Journal 16(2): 109-123.-   Andersen, D. C. and C. F. Goochee (1994). “The effect of    cell-culture conditions on the oligosaccharide structures of    secreted glycoproteins.” Current Opinion in Biotechnology 5:    546-549.-   Bardor, M., L. Faye, et al. (1999). “Analysis of the N-glycosylation    of recombinant glycoproteins produced in transgenic plants.” Trends    in Plant Science 4(9): 376-380.-   Bretthauer, R. K. and F. J. Castellino (1999). “Glycosylation of    Pichia pastoris-derived proteins.” Biotechnology and Applied    Biochemistry 30: 193-200.-   Burda, P. and M. Aebi (1999). “The dolichol pathway of N-linked    glycosylation.” Biochimica Et Biophysica Acta-General Subjects    1426(2): 239-257.-   Chiba, Y., M. Suzuki, et al. (1998). “Production of human compatible    high mannose-type (Man(5)GlcNAc(2)) sugar chains in Saccharomyces    cerevisiae.” Journal of Biological Chemistry 273(41): 26298-26304.-   Cole, E. S., E. Higgins, et al. (1994). “Glycosylation Patterns of    Human Proteins Expressed in Transgenic Goat Milk.” Journal of    Cellular Biochemistry: 265-265.-   Davies et al. Biotechnol Bioeng. 2001 Aug. 20; 74(4):288-294.    (Expression of GnTIII in a Recombinant Anti-CD20 CHO Production Cell    Line: Expression of Antibodies with Altered Glycoforms Leads to an    Increase in ADCC Through Higher Affinity for FcgRIII).-   Dente, L., U. Ruther, et al. (1988). “Expression of Human    Alpha-1-Acid Glycoprotein Genes in Cultured-Cells and in Transgenic    Mice.” Genes & Development 2(2): 259-266.-   Huffaker, T. C. and P. W. Robbins (1983). “Yeast Mutants Deficient    in Protein Glycosylation.” Proceedings of the National Academy of    Sciences of the United States of America-Biological Sciences 80(24):    7466-7470.-   Jarvis, D. L., Z. S. Kawar, et al. (1998). “Engineering    N-glycosylation pathways in the baculovirus-insect cell system.”    Current Opinion in Biotechnology 9(5): 528-533.-   Kimura, T., N. Kitamoto, et al. (1997). “A novel yeast gene, RHK1,    is involved in the synthesis of the cell wall receptor for the HM-1    killer toxin that inhibits beta-1,3-glucan synthesis.” Molecular &    General Genetics 254(2): 139-147.-   Kimura, T., T. Komiyama, et al. (1999). “N-glycosylation is involved    in the sensitivity of Saccharomyces cerevisiae to HM-1 killer toxin    secreted from Hansenula mrakii IFO 0895.” Applied Microbiology and    Biotechnology 51(2): 176-184.-   Malissard, M., S. Zeng, et al. (2000). “Expression of functional    soluble forms of human beta-1,4-galactosyltransferase I,    alpha-2,6-sialyltransferase, and alpha-1,3-fucosyltransferase VI in    the methylotrophic yeast Pichia pastoris.” Biochemical and    Biophysical Research Communications 267(1): 169-173.-   Maras, M. and R. Contreras (1994). Methods of Modifying Carbohydrate    Moieties. United States, Alko Group Ltd., Helsinki, Finland.-   Maras, M., A. De Bruyn, et al. (1999). “In vivo synthesis of complex    N-glycans by expression of human N-acetylglucosaminyltransferase I    in the filamentous fungus Trichoderma reesei.” Febs Letters 452(3):    365-370.-   Maras, M., X. Saelens, et al. (1997). “In vitro conversion of the    carbohydrate moiety of fungal glycoproteins to mammalian-type    oligosaccharides—Evidence for    N-acetylglucosaminyltransferase-1-accepting glycans from Trichoderma    reesei.” European Journal of Biochemistry 249(3): 701-707.-   Martinet, W., M. Maras, et al. (1998). “Modification of the protein    glycosylation pathway in the methylotrophic yeast Pichia pastoris.”    Biotechnology Letters 20(12): 1171-1177.-   McGarvey, P. B., J. Hammond, et al. (1995). “Expression of the    Rabies Virus Glycoprotein in Transgenic Tomatoes.” Bio-Technology    13(13): 1484-1487.-   Moens, S, and J. Vanderleyden (1997). “Glycoproteins in    prokaryotes.” Archives of Microbiology 168(3): 169-175.-   Nakanishishindo, Y., K. Nakayama, et al. (1993). “Structure of the    N-Linked Oligosaccharides That Show the Complete Loss of    Alpha-1,6-Polymannose Outer Chain From Och1, Och1 Mnn1, and Och1    Mnn1 Alg3 Mutants of Saccharomyces-Cerevisiae.” Journal of    Biological Chemistry 268(35): 26338-26345.-   Raju, T. S., J. B. Briggs, et al. (2000). “Species-specific    variation in glycosylation of IgG: evidence for the species-specific    sialylation and branch-specific galactosylation and importance for    engineering recombinant glycoprotein therapeutics.” Glycobiology    10(5): 477-486.-   Sharma, C. B., R. Knauer, et al. (2001). “Biosynthesis of    lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the    DoI-P-Man: Man(5)GlcNAc(2)-PP-DoI mannosyltransferase.” Biological    Chemistry 382(2): 321-328.-   Staub, J. M., B. Garcia, et al. (2000). “High-yield production of a    human therapeutic protein in tobacco chloroplasts.” Nature    Biotechnology 18(3): 333-338.-   Takeuchi, M. (1997). “Trial for molecular breeding of yeast for the    production of glycoprotein therapeutics.” Trends in Glycoscience and    Glycotechnology 9: S29-S35.-   Umana et al., Nat. Biotechnol. 1999a February (17)176-180.    (Engineered glycoforms of an antineuroblastoma IgG 1 with optimized    antibodydependent cellular cytotoxic activity)-   Umana et al., Biotechnol Bioeng. 1999b Dec. 5; 65(5):542-549.    (Regulated Overexpression of glycosyltransferase).-   Verostek, M. F., P. H. Atkinson, et al. (1993).    “Glycoprotein-Biosynthesis in the Alg3 Saccharomyces-Cerevisiae    Mutant .1. Role of Glucose in the Initial Glycosylation of Invertase    in the Endoplasmic-Reticulum.” Journal of Biological Chemistry    268(16): 12095-12103.-   Verostek, M. F., P. H. Atkinson, et al. (1993).    “Glycoprotein-Biosynthesis in the Alg3 Saccharomyces-Cerevisiae    Mutant .2. Structure of Novel Man6-10glenac2 Processing    Intermediates On Secreted Invertase.” Journal of Biological    Chemistry 268(16): 12104-12115.-   Weikert, S., D. Papac, et al. (1999). “Engineering Chinese hamster    ovary cells to maximize sialic acid content of recombinant    glycoproteins.” Nature Biotechnology 17(11): 1116-1121.-   Werner, R. G., W. Noe, et al. (1998). “Appropriate mammalian    expression systems for biopharmaceuticals.”    Arzneimittel-Forschung-Drug Research 48(8): 870-880.-   Yang, M. and M. Butler (2000). “Effects of ammonia on CHO cell    growth, erythropoietin production, and glycosylation.” Biotechnology    and Bioengineering 68(4): 370-380 Zufferey, R., R. Knauer, et al.    (1995). “Stt3, a Highly Conserved Protein Required for Yeast    Oligosaccharyl Transferase-Activity in-Vivo.” EMBO Journal 14(20):    4949-4960.

What is claimed is:
 1. A method for producing a human-like glycoproteinin a Pichia sp. host cell that does not display a 1,6mannosyltransferase activity with respect to the N-glycan on aglycoprotein, the method comprising the step of introducing into thehost cell a nucleic acid encoding a fusion protein comprising thecatalytic domain of a α-1,2-mannosidase fused at the N-terminus to anSEC12, VAN1, or MNN10 cellular targeting signal peptide.
 2. The methodclaim 1, wherein the host cell is capable of producing glycoproteinscomprising N-glycans, wherein at least 30 mole % of the N-glycans on theglycoprotein comprise a Man₅GlcNAc₂ carbohydrate structure.
 3. Themethod cell of claim 1, wherein the host cell is capable of producingglycoproteins comprising N-glycans, wherein at least 50 mole % of theN-glycans on the glycoprotein comprise a Man₅GlcNAc₂ carbohydratestructure.
 4. The method of claim 1, wherein the host cell comprises anucleic acid encoding a fusion protein comprising the catalytic domainof mouse mannosidase IA fused at the N-terminus to an SEC12 cellulartargeting signal peptide.
 5. The method of claim 1, wherein the hostcell comprises a nucleic acid encoding a fusion protein comprising thecatalytic domain of C. elegans mannosidase IB fused at the N-terminus toan VAN1 cellular targeting signal peptide.
 6. The method of claim 1,wherein the host cell comprises a nucleic acid encoding a fusion proteincomprising the catalytic domain of C. elegans mannosidase IB fused atthe N-terminus to an MNN10 cellular targeting signal peptide.
 7. Themethod of claim 1, wherein the host cell further expresses the catalyticdomains of one or more enzymes selected from the group consisting ofUDP-GlcNAc transferase, UDP-galactosyltransferase,GDP-fucosyltransferase, CMP-sialyltransferase, UP-GlcNAc transporter,UDP-galactose transporter, GDP-fucose transporter, CMP-sialic acidtransporter, and nucleotide diphosphatases.
 8. The method of claim 1,wherein the host cell further expresses GnTI and UDP-GlcNAc transporteractivities.
 9. The method of claim 1, wherein the host cell furtherexpresses a UDP- or GDP-specific diphosphatase activity.
 10. The methodof claim 1, wherein the host cell is capable of producing glycoproteinscomprising N-glycans, wherein the N-glycans on glycoprotein comprisesone or more sugars selected from the group consisting of GlcNAc,galactose, sialic acid, and fucose.
 11. The method of claim 1, whereinthe host cell is capable of producing glycoproteins comprisingN-glycans, wherein the N-glycans on glycoprotein comprise at least oneoligosaccharide branch comprising the structure NeuNAcGalGlcNAcMan. 12.The method of claim 1, wherein the host cell is selected from the groupconsisting of: Pichia pastoris, Pichia finlandica, Pichia trehalophila,Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolicamethanolica and Hansenula polymorpha.13. The method of claim 1, wherein the host cell is Pichia pastoris. 14.The method of claim 1, wherein the host cell is an ochl mutant of Pichiapastoris.
 15. The method of claim 2, wherein the host cell is Pichiapastoris.
 16. The method of claim 4, wherein the host cell is Pichiapastoris.
 17. The method of claim 5, wherein the host cell is Pichiapastoris.
 18. The method of claim 6, wherein the host cell is Pichiapastoris.